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Posts Tagged ‘ovarian cancer’


New Generation of Platinated Compounds to Circumvent Resistance

Curator/Writer: Stephen J. Williams, Ph.D.

Resistance to chemotherapeutic drugs continues to be a major hurdle in the treatment of neoplastic disorders, irregardless if the drug is a member of the cytotoxic “older” drugs or the cytostatic “newer” personalized therapies like the tyrosine kinase inhibitors.  For the platinatum compounds such as cisplatin and carboplatin, which are mainstays in therapeutic regimens for ovarian and certain head and neck cancers, development of resistance is often regarded as the final blow, as new options for these diseases have been limited.

Although there are many mechanisms by which resistance to platinated compounds may develop the purpose of this posting is not to do an in-depth review of this area except to refer the reader to the book   Ovarian Cancer and just to summarize the well accepted mechanisms of cisplatin resistance including:

  • Decreased cellular cisplatin influx
  • Increased cellular cisplatin efflux
  • Increased cellular glutathione and subsequent conjugation, inactivation
  • Increased glutathione-S-transferase activity (GST) and subsequent inactivation, conjugation
  • Increased γ-GGT
  • Increased metallothionenes with subsequent conjugation, inactivation
  • Increased DNA repair: increased excision repair
  • DNA damage tolerance: loss of mismatch repair (MMR)
  • altered cell signaling activities and cell cycle protein expression

Williams, S.J., and Hamilton, T.C. Chemotherapeutic resistance in ovarian cancer. In: S.C. Rubin, and G.P. Sutton (eds.), Ovarian Cancer, pp.34-44. Lippincott, Wilkins, and Williams, New York, 2000.

Also for a great review on clinical platinum resistance by Drs. Maritn, Hamilton and Schilder please see the following Clinical Cancer Research link here.

This curation represents the scientific rationale for the development of a new class of platinated compounds which are meant to circumvent mechanisms of resistance, in this case the loss of mismatch repair (MMR) and increased tolerance to DNA damage.

An early step in the production of cytotoxicity by the important anticancer drug cisplatin and its analog carboplatin is the formation of intra- and inter-strand adducts with tumor cell DNA 1-3. This damage triggers a cascade of events, best characterized by activation of damage-sensing kinases (reviewed in 4), p53 stabilization, and induction of p53-related genes involved in apoptosis and cell cycle arrest, such as bax and the cyclin-dependent kinase inhibitor p21waf1/cip1/sdi1 (p21), respectively 5,6. DNA damage significantly induces p21 in various p53 wild-type tumor cell lines, including ovarian carcinoma cells, and this induction is responsible for the cell cycle arrest at G1/S and G2/M borders, allowing time for repair 7,8.  DNA lesions have the ability of  to result in an opening of chromatin structure, allowing for transcription factors to enter 56-58.  Therefore the anti-tumoral ability of cisplatin and other DNA damaging agents is correlated to their ability to bind to DNA and elicit responses, such as DNA breaks or DNA damage responses which ultimately lead to cell cycle arrest and apoptosis.  Therefore either repair of such lesions, the lack of recognition of such lesions, or the cellular tolerance of such lesions can lead to resistance of these agents.

resistmech2

Mechanisms of Cisplatin Sensitivity and Resistance. Red arrows show how a DNA lesion results in chemo-sensitivity while the beige arrow show common mechanisms of resistance including increased repair of the lesion, effects on expression patterns, and increased inactivation of the DNA damaging agent by conjugation reactions

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mechPtresistance

 

 

Increased DNA Repair Mechanisms of Platinated Lesion Lead to ChemoResistance

 

DNA_repair_pathways

Description of Different Types of Cellular DNA Repair Pathways. Nucleotide Excision Repair is commonly up-regulated in highly cisplatin resistant cells

 

 

 

 

 

 

 

 

 

 

 

Loss of Mismatch Repair Can Lead to DNA Damage Tolerance

dnadamage tolerance

 

 

 

 

 

 

 

 

In the following Cancer Research paper Dr. Vaisman in the lab of Dr. Steve Chaney at North Carolina (and in collaboration with Dr. Tom Hamilton) describe how cisplatin resistance may arise from loss of mismatch repair and how oxaliplatin lesions are not recognized by the mismatch repair system.
Cancer Res. 1998 Aug 15;58(16):3579-85.

The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts.

Abstract

Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.

 

 

The following are slides I had co-prepared with my mentor Dr. Thomas C. Hamilton, Ph.D. of Fox Chase Cancer Center on DNA Mismatch Repair, Oxaliplatin and Ovarina Cancer.

edinborough2mmrtranslesion1

 

 

 

 

 

 

Multiple Platinum Analogs of Cisplatin (like Oxaliplatin )Had Been Designed to be Sensitive in MMR Deficient Tumors

edinborough2diffptanalogs

 

 

 

 

 

 

mmroxaliplatin

 

 

 

 

 

 

edinborough2ptanalogsresist

 

 

 

 

 

 

edinborough2relresistptanalogsdifflines

 

 

 

 

 

 

edinborough2msimlmh2refract

 

 

 

 

 

 

edinborough2gogoxaliplatintrial

 

 

 

 

 

 

 

Please see below video on 2015 Nobel Laureates and their work to elucidate the celluar DNA repair mechanisms.

Clinical genetics expert Kenneth Offit gives an overview of Lynch syndrome, a genetic disorder that can cause colon (HNPCC) and other cancers by defects in the MSH2 DNA mismatch repair gene. (View Video)

 

 

References

  1. Johnson, S. W. et al. Relationship between platinum-DNA adduct formation, removal, and cytotoxicity in cisplatin sensitive and resistant human ovarian cancer cells. Cancer Res 54, 5911-5916 (1994).
  2. Eastman, A. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology and Therapeutics 34, 155-166 (1987).
  3. Zhen, W. et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Molecular and Cellular Biology 12, 3689-3698 (1992).
  4. Durocher, D. & Jackson, S. P. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol 13, 225-231 (2001).
  5. el-Deiry, W. S. p21/p53, cellular growth control and genomic integrity. Curr Top Microbiol Immunol 227, 121-37 (1998).
  6. Ewen, M. E. & Miller, S. J. p53 and translational control. Biochim Biophys Acta 1242, 181-4 (1996).
  7. Gartel, A. L., Serfas, M. S. & Tyner, A. L. p21–negative regulator of the cell cycle. Proc Soc Exp Biol Med 213, 138-49 (1996).
  8. Chang, B. D. et al. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene 19, 2165-70 (2000).
  9. Davies, N. P., Hardman, L. C. & Murray, V. The effect of chromatin structure on cisplatin damage in intact human cells. Nucleic Acids Res 28, 2954-2958 (2000).
  10. Vichi, P. et al. Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP. Embo J 16, 7444-7456 (1997).
  11. Xiao, G. et al. A DNA damage signal is required for p53 to activate gadd45. Cancer Res 60, 1711-9 (2000).

Other articles in this Open Access Journal on ChemoResistance Include:

Cancer Stem Cells as a Mechanism of Resistance

An alternative approach to overcoming the apoptotic resistance of pancreatic cancer

Mutation D538G – a novel mechanism conferring acquired Endocrine Resistance causes a change in the Estrogen Receptor and Treatment of Breast Cancer with Tamoxifen

Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?

Nitric Oxide Mitigates Sensitivity of Melanoma Cells to Cisplatin

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

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Bisphosphonates and Bone Metastasis [6.3.1]

Curator: Stephen J. Williams, Ph.D.

bisophosphonates chemical

General Structure of Bisphosphonates

One of the hallmarks of advanced cancer is the ability to metastasize (tumor cells migrating from primary tumor and colonize in a different anatomical site in the body) and many histologic types of primary tumors have the propensity to metastasize to the bone. One of the frequent complications occurring from bone metastasis is bone fractures and severe pain associated with these cancer-associated bone fractures. An additional problem is cancer-associated hypercalcemia, which may or may not be dependent on bone-metastasis. The main humoral factor associated with cancer-related hypercalcemia is parathyroid hormone–related protein, which is produced by many solid tumors (Paget’s disease). Parathyroid hormone–related protein increases calcium by activating parathyroid hormone receptors in tissue, which results in osteoclastic bone resorption; it also increases renal tubular resorption of calcium {see (1) Bower reference for more information). This curation involves three areas:

  1. The Changing Views How Bone Remodeling Occurs
  2. Early Development of Agents that Alter Bone Remodeling and Early Use in Cancer Patients
  3. Recent Developments Regarding Use of Bisphosphonates in Cancer Patients

As there are numerous articles (1360; more than to manually curate) on “bone”, “metastasis” and “bisphosphonates” the following link is to a Pubmed search on the terms

http://www.ncbi.nlm.nih.gov/pubmed/?term=bone+metastasis+bisphosphonates

In addition there are subset searches to show use of bisphosphonates in common cancers and files given below with numbers of articles:

Search terms with Pubmed link # citations
bone metastasis bisphosphonates 1360
+ breast 559
+ prostate 349
+ colon 9
+ lung 222
  1. The Changing Views How Bone Remodeling Occurs

Bone remodeling (or bone metabolism) is a lifelong process where mature bone tissue is removed from the skeleton (a process called bone resorption) and new bone tissue is formed (a process called ossification or new bone formation). These processes also control the reshaping or replacement of bone following injuries like fractures but also micro-damage, which occurs during normal activity. Remodeling responds also to functional demands of the mechanical loading.

In the first year of life, almost 100% of the skeleton is replaced. In adults, remodeling proceeds at about 10% per year.[1]

An imbalance in the regulation of bone remodeling’s two sub-processes, bone resorption and bone formation, results in many metabolic bone diseases, such as osteoporosis. Two main types of cells are responsible for bone metabolism: osteoblasts (which secrete new bone), and osteoclasts (which break bone down). The structure of bones as well as adequate supply of calcium requires close cooperation between these two cell types and other cell populations present at the bone remodeling sites (ex. immune cells).[4] Bone metabolism relies on complex signaling pathways and control mechanisms to achieve proper rates of growth and differentiation. These controls include the action of several hormones, including parathyroid hormone (PTH), vitamin D, growth hormone, steroids, and calcitonin, as well as several bone marrow-derived membrane and soluble cytokines and growth factors (ex. M-CSF, RANKL, VEGF, IL-6 family…). It is in this way that the body is able to maintain proper levels of calcium required for physiological processes.

Subsequent to appropriate signaling, osteoclasts move to resorb the surface of the bone, followed by deposition of bone by osteoblasts. Together, the cells that are responsible for bone remodeling are known as the basic multicellular unit (BMU), and the temporal duration (i.e. lifespan) of the BMU is referred to as the bone remodeling period.

For a good review on bone remodeling please see Bone remodelling in a nutshell

boneremodelPTHumich

bone remodeling 3

  1. Early Development of Agents that Alter Bone Remodeling and Early Use in Cancer Patients

Bisphosphonates had been first synthesized in the late 1800’s yet their development and approval for the indication of osteoporosis occurred over 100 years later, in the 1990’s. For a good review on the history of bisphosphonates please see the following review:

Historical perspectives on the clinical development of bisphosphonates in the treatment of bone diseases. Francis MD1, Valent DJ. J Musculoskelet Neuronal Interact. 2007 Jan-Mar;7(1):2-8.

For a good reference on bisphosphonates as a class, as well as indication, contraindication and side effects see University of Washington web page at http://courses.washington.edu/bonephys/opbis.html

 

Please view slideshow in the following link: The Evolving Role of Bisphosphonates for Cancer Treatment-Induced Bone Loss presentation by Richard L. Theriault, DO, MBA at MD Anderson Cancer Center

bisphosphonatecancerslide1

  1. Recent Developments Regarding Use of Bisphosphonates in Cancer Patients

Bone Metastasis Treatment with Bisphosphonates; A review form OncoLink

Source: From University of Pennsylvania OncoLink® at http://www.oncolink.org/types/article.cfm?c=708&id=9629

Julia Draznin Maltzman, MD and Modified by Lara Bonner Millar, MD
The Abramson Cancer Center of the University of Pennsylvania
Last Modified: December 18, 2014

Introduction

Bone metastases are a common complication of advanced cancer. They are especially prevalent (up to 70%) in breast and prostate cancer. Bone metastases can cause severe pain, bone fractures, life-threatening electrolyte imbalances, and nerve compression syndromes. The pain and neurologic dysfunction may be difficult to treat and significantly compromises the patients’ quality of life. Bone metastases usually signify advanced, often incurable disease.

Osteolytic vs. osteoblastic

Bony metastases are characterized as being either osteolytic or osteoblastic. Osteolytic means that the tumor caused bone break down or dissolution. This usually results in loss of calcium from bone. On X-rays these are seen as holes called “lucencies” within the bone. Diffuse osteolytic lesions are most characteristic of a blood cancer called Multiple Myeloma, however they may be present in patients with many other types of cancer.

Osteoblastic bony lesions, by contrast, are characterized by increased bone production. The tumor somehow signals to the bone to overproduce bone cells and result in rigid, inflexible bone formation. The cancer that typically causes osteoblastic bony lesions is prostate cancer. Most cancers result in either osteolytic or osteoblastic bony changes, but some malignancies can lead to both. Breast cancer patients usually develop osteolytic lesions, although at least 15-20 percent can have osteoblastic pathology.

Why the bone?

The bone is a common site of metastasis for many solid tissue cancers including prostate, breast, lung, kidney, stomach, bladder, uterus, thyroid, colon and rectum. Researchers speculate that this may be due to the high blood flow to the bone and bone marrow. Once cancer cells gain access to the blood vessels, they can travel all over the body and usually go where there is the highest flow of blood. Furthermore, tumor cells themselves secrete adhesive molecules that can bind to the bone marrow and bone matrix. This molecular interaction can cause the tumor to signal for increased bone destruction and enhance tumor growth within the bone. A recent scientific discovery showed that the bone is actually a rich source of growth factors. These growth factors signal cells to divide, grow, and mature. As the cancer attacks the bone, these growth factors are released and serve to further stimulate the tumor cells to grow. This results in a self-generating growth loop.

What are the symptoms of bone metastasis?

It must be recognized that the symptoms of bone metastasis can mimic many other disease conditions. Most people with bony pain do not have bone metastasis. That being noted, the most common symptom of a metastasis to the bone is pain. Another common presentation is a bone fracture without any history of trauma. Fracture is more common in lytic metastases than blastic metastases.

Some people with more advanced disease may come to medical attention because of numbness and tingling sensation in their feet and legs. They may have bowel and bladder dysfunction – either losing continence to urine and/or stool, or severe constipation and urinary retention. Others may complain of leg weakness and difficulty moving their legs against gravity. This would imply that there is tumor impinging on the spinal cord and compromising the nerves. This is considered an emergency called spinal cord compression, and requires immediate medical attention. Another less common presentation of metastatic disease to the bone is high levels of calcium in the body. High calcium can make patients constipated, result in abdominal pain, and at very high levels, can lead to confusion and mental status changes.

Diagnosis of bone metastasis

Once a patient experiences any of the symptoms of bone metastasis, various tests can be done to find the true cause. In some cases bone metastasis can be detected before the symptoms arise. X-rays, bone scans, and MRIs are used to diagnose this complication of cancer. X-rays are especially helpful in finding osteolytic lesions. These often appear as “holes” or dark spots in the bone on the x-ray film. Unfortunately, bone metastases often do not show up on plain x-rays until they are quite advanced. By contrast, a bone scan can detect very early bone metastases. This test is done by injecting the patient with a small amount of radio-tracing material in the vein. Special x-rays are taken sometime after the injection. The radiotracer will preferentially go to the site of disease and will appear as a darker, denser, area on the film. Because this technique is so sensitive, sometimes infections, arthritis, and old fractures can appear as dark spots on the bone scan and may be difficult to differentiate from a true cancer. Bone scans are also used to follow patients with known bone metastasis. Sometimes CT scan images can show if a cancer has spread to the bone. An MRI is most useful when examining nerve roots suspected of being compressed by tumor or bone fragments due to tumor destruction. It is used most often in the setting of spinal cord compromise.

There are no real blood tests that are currently used to diagnose a bone metastasis. There are, however, a number of blood tests that a provider can obtain that may suggest the presence of bone lesions, but the diagnosis rests with the combination of radiographic evidence, clinical picture, and natural history of the malignancy. For example, elevated levels of calcium or an enzyme called alkaline phosphatase can be related to bone metastasis, but these lab tests alone are insufficient to prove their presence.

Treatment

The best treatment for bony metastasis is the treatment of the primary cancer. Therapies may include chemotherapy, hormone therapy, radiation therapy, immunotherapy, or treatment with monoclonal antibodies. Pain is often treated with narcotics and other pain medications, such as non-steroidal anti-inflammatory agents. Physical therapy may be helpful and surgery may have an important role if the cancer resulted in a fracture of the bone.

Bisphosphonates

Bisphosphonates are s category of medications that decrease pain from bone metastasis and may improve overall bone health. Bisphosphonates man-made versions of a naturally occurring compound called pyrophosphate that prevents bone breakdown. They are a class of medications widely used in the treatment and prevention of osteoporosis and certain other bone diseases (such as Paget’s Disease), as well as in the treatment of elevated blood calcium. These drugs suppress bone breakdown by cells called osteoclasts, and, can indirectly stimulate the bone forming cells called osteoblasts. It is for this reason, and for the fact that bisphosphonates are very effective in relieving bone pain associated with metastatic disease, that they have transitioned to the oncology arena. However, treatment of bone metastases is not curative. There is increasing evidence that bisphosphonates can prevent bony complications in some metastatic cancers and may even improve survival in some cancers. Most researchers agree that these drugs are more helpful in osteolytic lesions and less so in osteoblastic metastasis in terms of bone restoration and health, but the bisphosphonates are able to alleviate pain associated with both types of lesions. The appropriate time to start treatment is once a bone metastasis has been identified on imaging.

Bisphosphonates can be given either orally or intravenously. The latter is the preferred route of administration for many oncologists as it is given monthly as a short infusion and does not have the gastrointestinal side effects that the oral bisphosphonates have. There are currently two approved and commonly used IV bisphosphonates –Pamidronate disodium (Aredia, Novartis) and zolendronic acid (Zometa, Novartis). Their side effect profile is fairly mild and includes a flu-like reaction during the first 48 hours after the infusion, kidney impairment and osteonecrosis of the jaw with long term use. Patients with renal impairment may not be candidates for this therapy.

Bisphophonates may have some level of anti-tumor activity in breast cancer. A recent Phase III clinical trial revealed that the addition of Zometa to endocrine therapy, improves disease-free survival, but not overall survival, in pre-menopausal patients with estrogen-receptor postive early breast cancer. Another trial called AZURE found no effect from the bisphosphonate zolendronic acid (Zometa, Novartis) on the recurrence of breast cancer or on overall survival. However, several other studies on bisphosphonates and breast cancer are ongoing, and for now, their use is not recommended in patients without metastases.

In addition to bisphosphonates, osteoclast inhibition can also be achieved through other means. Another medication, Denosumab (XGEVA, Amgen), targets a receptor called receptor activator of nuclear factor kappa B ligand (RANKL), is able to block osteoclast formation. A few studies comparing Denosumab to bisphosphonates have found Denosumab results in a longer time to skeletal events, on the order of a few months, compared to bisphosphonates, however many experts believe that the evidence is not strong enough to support one class of drug over another. The most common side effects of Denosumab are fatigue or asthenia, hypophosphatemia, hypocalcemia and nausea. Patients receiving bisphosphonates or denosumab should also be taking calcium and vitamin D supplementation.

The future

Skeletal metastases remain one of the more debilitating problems for cancer patients. Research is ongoing to identify the molecular mechanisms that result in both osteolytic and osteoblastic bone lesions. Perhaps the use of proteomics and gene array data may permit us to identify some factors specific to the tumor or to the bony lesion itself that could be used as therapeutic targets to teat or even prevent this complication.

In summary

  •  there is well established evidence in preclinical models that bisphosphonates:reduce the total tumor burden in bone
  • it is unclear as to the mechanisms of this preclinical finding as bisphosphonates have been shown to directly have antitumor activity
  • as the review by Holen I1, Coleman RE.show “Bisphosphonates as treatment of bone metastases” (abstract given below) there is conflicting clinical evidence of this effect found in preclinical models

Accelerated bone loss is a common clinical feature of advanced breast cancer, and anti-resorptive bisphosphonates are the current standard therapy used to reduce the number and frequency of skeletal-related complications experienced by patients. Bisphosphonates are potent inhibitors of bone resorption, acting by inducing osteoclast apoptosis and thereby preventing the development of cancer-induced bone lesions. In clinical use bisphosphonates are mainly considered to be bone-specific agents, but anti-tumour effects have been reported in a number of in vitro and in vivo studies. By combining bisphosphonates with chemotherapy agents, growth and progression of breast cancer bone metastases can be virtually eliminated in model systems. Recent clinical trials have indicated that there may be additional benefits from bisphosphonate treatment, including positive effects on recurrence and survival when added to standard endocrine therapy. Whereas the ability of bisphosphonates to reduce cancer-induced bone disease is well established, their potential direct anti-tumour effect remain controversial. Ongoing clinical trials will establish whether bisphosphonates can inhibit the development of bone metastases in high-risk breast cancer patients. This review summarizes the main studies that have investigated the effects of bisphosphonates, alone and in combination with other anti-cancer agents, using in vivo model systems of breast cancer bone metastases. We also give an overview of the use of bisphosphonates in the treatment of breast cancer, including examples of key clinical trials. The potential side effects and future clinical applications of bisphosphonates will be outlined.

References

  1. Bower M, Cox S. Endocrine and metabolic complications of advanced cancer. In: Doyle D, Hanks G, Cherny NI, Calman K, editors. Oxford textbook of palliative medicine. 3rd ed. New York, NY: Oxford University Press; 2004. p. 688-90.

Henry DH, Costa L, Goldwasser F, et al. Randomized, double-blind study of denosumab versus zoledronic acid in the treatment of bone metastases in patients with advanced cancer (excluding breast and prostate cancer) or multiple myeloma. J Clin Oncol. 2011;29(9):1125-32.

Van Poznak CH, Temin S, Yee GC, et al. American Society of Clinical Oncology executive summary of the clinical practice guideline update on the role of bone-modifying agents in metastatic breast cancer. J Clin Oncol. 2011;29(9):1221-7.

West, H. Denosumab for prevention of skeletal-related events in patients with bone metastases from solid tumors: incremental benefit, debatable value. J Clin Oncol. 2011;29(9):1095-8.

Gnant M, Mlineritsch B, Schippinger W et al.: Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med. 360(7),679–691 (2009).

Treatment Guidelines by Cancer Organizations

ASCO Issues Updated Guideline on the Role of Bone-Modifying Agents in the Prevention and Treatment of Bone Metastases in Patients with Metastatic Breast Cancer

For Immediate Release

February 22, 2011

Contact:

Steven Benowitz
571-483-1370
steven.benowitz@asco.org

ALEXANDRIA, Va. – The American Society of Clinical Oncology (ASCO) today issued an update to its clinical practice guideline on the use of bone-modifying agents, in particular, osteoclast inhibitors, to prevent and treat skeletal complications from bone metastases in patients with metastatic breast cancer. The new guideline includes recommendations on the use of a new drug option, denosumab (Xgeva), and addresses osteonecrosis of the jaw, an uncommon condition that may occur in association with bone-modifying agents. The updated guideline also provides new recommendations on monitoring of patients who undergo treatment with bone-modifying agents and highlights priorities for future research on these drugs.

ASCO’s Bisphosphonates in Breast Cancer Panel conducted a systematic review of the medical literature to develop the new recommendations. The updated guideline, American Society of Clinical Oncology Clinical Practice Guideline Update on the Role of Bone-Modifying Agents in Metastatic Breast Cancer, was published online today in the Journal of Clinical Oncology.

The guideline recommends that patients with breast cancer who have evidence of bone metastases be given one of three agents – denosumab, pamidronate or zoledronic acid – approved by the U.S. Food and Drug Administration. It does not support use of any one drug over the others. These drugs are all considered osteoclast inhibitors, but they belong to different drug families: pamidronate and zoledronic acid are part of a class of drugs called bisphosphonates, while denosumab is a monoclonal antibody that targets receptor activator of nuclear factor-kappa beta ligand (RANKL).

The guideline also recommends against initiating bone-modifying agents in the absence of bone metastases outside of a clinical trial. It notes that an abnormal bone scan result alone, without confirmation by a radiograph, CT or MRI scan, is not sufficient evidence to support treatment with these drugs.

“The updated recommendations take into account recent progress in controlling potential bone damage in metastatic breast cancer,” said Catherine Van Poznak, MD, co-chair of the Bisphosphonates in Breast Cancer Panel and assistant professor of medicine at the University of Michigan. “We’ve established that a growing number of osteoclast inhibitors can have a positive effect and decrease of the risk of skeletal-related events in women with bone metastases. Because many factors – including medical and economic – must be considered when selecting a therapy for an individual, it’s good to have several effective choices.”

Bone is one of the most common sites to which breast cancer spreads. Bone metastases occur in approximately 70 percent of patients with metastatic disease. These metastases can cause bone cells (osteoclasts) to become overactive, which can result in excessive bone loss, disrupting the bone architecture and causing skeletal-related events (SREs), such as fracture, the need for surgery or radiation therapy to bone, spinal cord compression and hypercalcemia of malignancy.

This document updates guideline recommendations that were first issued in 2000 and revised in 2003, and focused on the use of bisphosphonates. The current guideline uses the more inclusive term, bone-modifying agents, to reflect a wider category of therapeutic agents such as monoclonal antibodies that use different mechanisms of action to prevent and treat damage from bone metastases. The guideline notes that research remains to be conducted to address several areas where questions remain.

“The guideline considers new data in a variety of areas, including studies showing that denosumab has equivalent effectiveness compared with other currently available drug therapies,” explained bisphosphonates panel co-chair Jamie Von Roenn, MD, professor of medicine at Northwestern University. “The guideline also provides guidance on preventing a rare, but significant complication of therapy with bone-modifying agents, osteonecrosis of the jaw.”

Denosumab is a human monoclonal antibody that targets a receptor, RANKL, involved in the regulation of bone remodeling. The guideline cites evidence from a randomized Phase III trial showing that denosumab appears to be comparable to zoledronic acid in reducing the risk of SREs in women with bone metastases from breast cancer. Denosumab is given subcutaneously, and can have side effects such as hypocalcemia.

The guideline also addresses the recently discovered osteonecrosis of the jaw. The first reports of this degenerative condition were published in the medical and dental literature in 2003. The committee recommended that all patients with breast cancer get dental evaluations and receive preventive dentistry care before beginning treatment with bone-modifying osteoclast inhibitors.

The panel updated its recommendations regarding the effects of bisphosphonates on kidney function, particularly for those taking either pamidronate or zoledronic acid, which have been associated with deteriorating kidney function. It said that clinicians should monitor serum creatinine clearance prior to each dose of pamidronate or zoledronic acid according to FDA-approved labeling.

The panel did not recommend using biochemical markers to monitor bone-modifying agent effectiveness and use outside of a clinical trial.

While many of the 2003 recommendations remain the same, the guideline notes several research directions to be addressed, including:

  • Duration of therapy with bone modifying agents, and the timing or intervals between delivery.
  • The development of a risk index for SREs, and better ways to stratify patient risk of SRE or risk of toxicity from a bone-modifying agent. Individual risk may guide selection of timing for use of a bone-modifying agent therapy.
  • Trials specifically examining whether stage IV breast cancer patients who do not have evidence of bone metastases would benefit from bone-modifying agents.
  • The role of biomarkers in treatment selection and monitoring drug effectiveness.
  • Understanding the optimal dosing of calcium and vitamin D supplementation in patients treated with bone-modifying agents.

The meta-analysis from the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) was published in Lancet and suggested that “Adjuvant bisphosphonates reduce the rate of breast cancer recurrence in the bone and improve breast cancer survival, but there is definite benefit only in women who were postmenopausal when treatment began”.

Results

  • Of 18, 206 women in trials of 2-5 years of bisphosphonate3453 first recurrences, and 2106 subsequent deaths.
  • Overall, the reductions in recurrence (RR 0·94, 95% CI 0·87-1·01; 2p=0·08), distant recurrence (0·92, 0·85-0·99; 2p=0·03), and breast cancer mortality (0·91, 0·83-0·99; 2p=0·04) were of only borderline significance
  • Among premenopausal women, treatment had no apparent effect on any outcome, but among 11 767 postmenopausal women it produced highly significant reductions in recurrence (RR 0·86, 95% CI 0·78-0·94; 2p=0·002), distant recurrence (0·82, 0·74-0·92; 2p=0·0003), bone recurrence (0·72, 0·60-0·86; 2p=0·0002), and breast cancer mortality (0·82, 0·73-0·93; 2p=0·002). “This was iregardless of age or bisphosphonate type.

Lancet. 2015 Jul 23. pii: S0140-6736(15)60908-4. doi: 10.1016/S0140-6736(15)60908-4. Adjuvant bisphosphonate treatment in early breast cancer: meta-analyses of individual patient data from randomised trials.

Early Breast Cancer Trialists’ Collaborative Group (EBCTCG).

This Study was reported at the 36th Annual San Antonio Breast Cancer Symposium (SABCS): Abstract S4-07. Presented December 12, 2013 and Medscape Medical News journalist Kate Johnson covered the finding with author interviews in the following article:

Bisphosphonates: ‘New Addition’ to Breast Cancer Treatment?

Kate Johnson

December 13, 2013

Editors’ Recommendations

SAN ANTONIO — Adjuvant bisphosphonate treatment significantly improves breast cancer survival and reduces bone recurrence in postmenopausal women with early breast cancer, according to a meta-analysis reported here at the 36th Annual San Antonio Breast Cancer Symposium.

“We have finally defined a new addition to standard treatment,” announced lead investigator Robert Coleman, MD, professor of oncology at the University of Sheffield in the United Kingdom. He emphasized that, as hypothesized, the benefits of this therapy were confined to postmenopausal women.

“There is absolutely no effect on mortality in premenopausal women, with a hazard ratio [HR] of 1.0,” he reported. “But for postmenopausal women, we see a 17% reduction in the risk of death [HR, 0.83], which is highly statistically significant.”

In terms of the absolute benefit, bisphosphonates decreased the breast cancer mortality rate from 18.3% to 15.2% in postmenopausal women (P = .004).

The separation of benefit by menopausal status was also seen in the bone recurrence data.

In premenopausal women, there is no significant effect on bone recurrence (HR, 0.93), whereas in postmenopausal women, there was a 34% reduction. The difference was “highly significant,” said Dr. Coleman.

“I personally believe adjuvant bisphosphonates should be standard treatment in postmenopausal women with breast cancer,” said Michael Gnant, MD, professor of surgery at the Medical University of Vienna, who was one of the study investigators. He spoke during a plenary session before the results were formally announced. (Please click this LINK to See VIDEO Interview with Dr. Gnant)

“This is an important analysis,” said Rowan Chlebowski, MD, PhD, medical oncologist from the Harbor-UCLA Medical Center in Los Angeles.

“There will be a substantial increase in the use of bisphosphonates,” he told Medscape Medical News after the presentation.

“The only question is whether people will accept this analysis as the final word.” Dr. Chlebowski explained that some people might criticize the study as being a post hoc analysis of previous findings.

“You might find some mixed feelings about whether this should be accepted, but I think this will get people thinking,” he said. Dr. Chlebowski previously reported a large observational study that demonstrated that postmenopausal women taking oral bisphosphonates for osteoporosis had a significantly lower risk for breast cancer.

Bisphosphonates were originally indicated for the treatment of osteoporosis, and include agents such as alendronate (Fosamax, Merck), ibandronate (Boniva, Genentech), risedronate (Actonel, sanofi-aventis), and zoledronic acid (Reclast, Novartis). But they are also indicated for bone-related use in breast cancer patients, Dr. Chlebowski pointed out.

Because bisphosphonates “also have an indication for preventing bone loss associated with aromatase inhibitor use, they are already approved in this setting, and would prevent recurrences. It will be interesting to see if guideline panels” like these findings, he noted.

Why Postmenopausal Women Benefit

In the plenary session, Dr. Gnant acknowledged that the data on bisphosphonates to date have been mixed.

There are “many trials showing controversial results” for bisphosphonates in the context of breast cancer, he said. “When we put them all together in an unselected population, some show beneficial effects and some do not.”

Dr. Gnant explained why bisphosphonates appear to be effective in older but not younger women. “When you confine your analysis to the low-estrogen environment, postmenopausal women, or women rendered menopausal by ovarian function suppression, we see that all these trials show a consistent benefit for these patients,” he said.

“Essentially, this low-estrogen hypothesis as a prerequisite for adjuvant bisphosphonate activity means that we believe these treatments can silence the bone marrow microenvironment. However, this only translates to relevant clinical benefits in low-estrogen environments,” he added.

More Study Details

The meta-analysis involved 36 trials of adjuvant bisphosphonates in breast cancer with 17,791 pre- and postmenopausal women.

The primary outcomes of the study were time to distant recurrence, local recurrence, and new second primary breast cancer (ipsilateral or contralateral), time to first distant recurrence (ignoring any previous locoregional or contralateral recurrences), and breast cancer mortality.

Planned subgroup analyses based on hypotheses generated from previous findings included site of recurrence, site of first distant metastasis, menopausal status, and type and schedule of bisphosphonate therapy, said Dr. Coleman.

With bisphosphonate therapy, there was a nonsignificant 1% reduction in breast cancer recurrence at 10 years in postmenopausal women, compared with premenopausal women (25.4% vs 26.5%), and “a small borderline advantage” for distant recurrence (20.9% vs 22.3%), he reported.

However, there was a significant benefit of bisphosphonates in bone recurrence in postmenopausal women (6.9% vs 8.4%; P = .0009), with no effect on nonbone recurrence.

There was no impact of bisphosphonates on local recurrence or cancer in the contralateral breast.

For distant recurrence, there was a 3.5% absolute benefit in postmenopausal women (18.4% vs 21.9%; P = .0003); for distant recurrence, there is was a significant improvement of 2.9% in bone recurrence (5.9% vs 8.8%; P < .00001).

There was no significant reduction in first distant recurrence outside bone, and risk reductions were similar, irrespective of estrogen-receptor status, node status, or use or not of chemotherapy.

“Adjuvant bisphosphonates reduce bone metastases and improve survival in postmenopausal women,” concluded Dr. Coleman. “We have statistical security in this result, with a 34% reduction in the risk of bone recurrence (P = .00001), and a 17% — or 1 in 6 — reduction in the risk of breast cancer death (P =.004).”

The analysis struck a clear line between pre- and postmenopausal women — something that was revealed in a subgroup analysis the AZURE trial, which Dr. Coleman was involved in (N Engl J Med. 2011;365:1396-1405).

Because of this, he was asked about the validity of basing the current analysis on the AZURE hypothesis-generating population.

“We repeated the analysis without the AZURE patients, because they are the hypothesis-generating population, and the P values and risk reductions did not change,” he explained.

Source: Medscape Medical News at http://www.medscape.com/viewarticle/817787#vp_1

Updated on 10/20/2015: Other articles for reference on Bisphosphonates and Metastasis

Clin Exp Metastasis. 2015 Oct;32(7):689-702. doi: 10.1007/s10585-015-9737-y. Epub 2015 Aug 1.

Human breast cancer bone metastasis in vitro and in vivo: a novel 3D model system for studies of tumour cell-bone cell interactions.

Author information

  • 1Academic Unit of Clinical Oncology, Department of Oncology, Mellanby Centre for Bone Research, Medical School, University of Sheffield, Sheffield, S10 2RX, UK.
  • 2Department of Human Metabolism, Mellanby Centre for Bone Research, Medical School, University of Sheffield, Sheffield, S10 2RX, UK.
  • 3Academic Unit of Clinical Oncology, Department of Oncology, Mellanby Centre for Bone Research, Medical School, University of Sheffield, Sheffield, S10 2RX, UK. p.d.ottewell@sheffield.ac.uk.

Abstract

Bone is established as the preferred site of breast cancer metastasis. However, the precise mechanisms responsible for this preference remain unidentified. In order to improve outcome for patients with advanced breast cancer and skeletal involvement, we need to better understand how this process is initiated and regulated. As bone metastasis cannot be easily studied in patients, researchers have to date mainly relied on in vivo xenograft models. A major limitation of these is that they do not contain a human bone microenvironment, increasingly considered to be an important component of metastases. In order to address this shortcoming, we have developed a novel humanised bone model, where 1 × 10(5) luciferase-expressing MDA-MB-231 or T47D human breast tumour cells are seeded on viable human subchaodral bone discs in vitro. These discs contain functional osteoclasts 2-weeks after in vitro culture and positive staining for calcine 1-week after culture demonstrating active bone resorption/formation. In vitro inoculation of MDA-MB-231 or T47D cells colonised human bone cores and remained viable for <4 weeks, however, use of matrigel to enhance adhesion or a moving platform to increase diffusion of nutrients provided no additional advantage. Following colonisation by the tumour cells, bone discs pre-seeded with MDA-MB-231 cells were implanted subcutaneously into NOD SCID mice, and tumour growth monitored using in vivo imaging for up to 6 weeks. Tumour growth progressed in human bone discs in 80 % of the animals mimicking the later stages of human bone metastasis. Immunohistochemical and PCR analysis revealed that growing MDA-MB-231 cells in human bone resulted in these cells acquiring a molecular phenotype previously associated with breast cancer bone metastases. MDA-MB-231 cells grown in human bone discs showed increased expression of IL-1B, HRAS and MMP9 and decreased expression of S100A4, whereas, DKK2 and FN1 were unaltered compared with the same cells grown in mammary fat pads of mice not implanted with human bone discs.

Cancer. 2000 Jun 15;88(12 Suppl):2979-88.

Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.

Abstract

BACKGROUND:

Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis.

METHODS:

The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney.

RESULTS:

BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4T1 mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma.

CONCLUSIONS:

These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases.

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Hormone Therapy [9.6]

Writer and Curator: Larry H. Bernstein, MD, FCAP

The structure of this article is as follows:

9.6.1 Hormone Treatment Fights Prostate Cancer

9.6.2 Diabetes and Cardiovascular Disease During Androgen Deprivation Therapy for Prostate Cancer

9.6.3 Breast Cancer and Hormone Therapy

9.6.4 Hormone Therapy and Different Ovarian Cancers

9.6.5 Chemotherapy versus hormonal treatment in platinum- and paclitaxel-refractory ovarian cancer: a randomised trial of the German Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) Study Group Ovarian Cancer

Introduction

9.6.1 Hormone Treatment Fights Prostate Cancer

By R. Morgan Griffin
http://www.webmd.com/prostate-cancer/features/hormone-therapy-for-prostate-cancer

Hormone therapy for prostate cancer has come a long way in the past few decades. Not so long ago, the only hormonal treatment for this disease was drastic: an orchiectomy, the surgical removal of the testicles.

Now we have a number of medications — available as pills, injections, and implants — that can give men the benefits of decreasing male hormone levels without irreversible surgery.

“I think hormonal therapy has done wonders for men with prostate cancer,” Stuart Holden, MD, Medical Director of the Prostate Cancer Foundation.

Hormone therapy for prostate cancer does have limitations. Right now, it’s usually used only in men whose cancer has recurred or spread elsewhere in the body.

But even in cases where removing or killing the cancer isn’t possible, hormone therapy can help slow down cancer growth. Though it isn’t a cure, hormone therapy for prostate cancer can help men with prostate cancer feel better and add years to their lives.

On average, hormone therapy can stop the advance of cancer for two to three years. However, it varies from case to case. Some men do well on hormone therapy for much longer.

The idea that hormones have an effect on prostate cancer is not new. The scientist Charles Huggins first established this over 60 years ago in work that led to his winning the Nobel Prize. Huggins found that removing one of the main sources of male hormones from the body — the testicles — could slow the growth of the disease.

“This procedure worked dramatically,” says Holden, who is also director of the Prostate Cancer Center at Cedar Sinai Medical Center in Los Angeles. “Before, these men were confined to bed and wracked with pain. Almost immediately afterwards, they improved.”

Huggins found that some types of prostate cancer cells androgens — to grow. Testosterone is one kind of androgen. About 90% to 95% of all androgens are made in the testicles, while the rest are made in the adrenal glands.

Hormone therapy for prostate cancer works by either preventing the body from making these androgens or by blocking their effects. Either way, the hormone levels drop, and the cancer’s growth slows.

In 85% to 90% of cases of advanced prostate cancer, hormone therapy can shrink the tumor.

However, hormone therapy for prostate cancer doesn’t work forever. The problem is that not all cancer cells need hormones to grow. Over time, these cells that aren’t reliant on hormones will spread. If this happens, hormone therapy won’t help anymore, and your doctor will need to shift to a different treatment approach.

There are two basic kinds of hormone therapy for prostate cancer. One class of drugs stops the body from making certain hormones. The other allows the body to make these hormones, but prevents them from attaching to the cancer cells. Some doctors start treatment with both drugs in an effort to achieve a total androgen block. This approach goes by several names: combined androgen blockade, complete androgen blockade, or total androgen blockade.

Here’s a rundown of the techniques.

  • Luteinizing hormone-releasing hormone agonists (LHRH agonists.)These are chemicals that stop the production of testosterone in the testicles. Essentially, they provide the benefits of an orchiectomy for men with advanced prostate cancer without surgery. This approach is sometimes called “chemical castration.” However, the effects are fully reversible if you stop taking the medication.Most LHRH agonists are injected every one to four months. Some examples are Lupron, Trelstar, Vantas, and Zoladex. A new drug, Viadur, is an implant placed in the arm just once a year.

    Side effects can be significant. They include: loss of sex drivehot flashes, development of breasts (gynecomastia) or painful breasts, loss of muscle, weight gain, fatigue, and decrease in levels of “good”cholesterol.

    Plenaxis is a drug that’s similar to LHRH agonists. However, because it can cause serious allergic reactions, it’s not used that often.

  • Anti-androgens. LHRH agonists and orchiectomies only affect the androgens that are made in the testicles. Thus they have no effect on the 5% to 10% of a man’s “male” hormones that are made in the adrenal glands. Anti-androgens are designed to affect the hormones made in the adrenal glands. They don’t stop the hormones from being made, but they stop them from having an effect on the cancer cells.The advantage of anti-androgens is that they have fewer side effects than LHRH agonists. Many men prefer them because they are less likely to diminish libido. Side effects include tenderness of the breasts, diarrhea, and nausea. These drugs are also taken as pills each day, which may be more convenient than injections. Examples are CasodexEulexin, and Nilandron.

    In some cases, starting treatment with an LHRH agonist can cause a “tumor flare,” a temporary acceleration of the cancer’s growth due to an initial increase in testosterone before the levels drop. This may cause the prostate gland to enlarge, obstructing the bladder and making it difficult to urinate. It’s believed that starting with an anti-androgen drug and then switching to an LHRH agonist can help avoid this problem. In patients with bone metastases, this “flare” can lead to significant complications such as bone pain, fractures, and nerve compression.

    Strangely, if treatment with an anti-androgen doesn’t work, stopping it may actually improve symptoms for a short time. This phenomenon is called “androgen withdrawal,” and experts aren’t sure why it happens.

  • Combined Androgen Blockade. This approach combines anti-androgens with LHRH agonists or an orchiectomy. By using both approaches, you can cut off or block the effects of hormones made by both the adrenal glands and the testicles. However, using both treatments can also increase the side effects. An orchiectomy or an LHRH agonist on its own can cause significant side effects like a loss of libido, impotence, and hot flashes. Adding an anti-androgen can cause diarrhea, and less often, nauseafatigue, and liver problems.
  • Estrogens. Some synthetic versions of female hormones are used for prostate cancer. In fact, they were one of the early treatments used for the disease. However, because of their serious cardiovascular side effects, they’re not used as often anymore. J. Brantley Thrasher, MD, a spokesman for the American Urological Association and chairman of urology at the University of Kansas Medical Center, says they’re usually used only after initial hormone treatments have failed. Examples of estrogens are DES (diethylstilbestrol), Premarin, and Estradiol.
  • Other Drugs. Proscar (finasteride) is another drug that indirectly blocks an androgen that helps prostate cancer cells grow. Depending

on the case, doctors sometimes use other anticancer drugs like Nizoral (ketoconazole) and Cytadren (aminoglutethimide.)

  • Orchiectomy. The surgical removal of the testicles was the earliest form of hormone therapy for prostate cancer. However, the procedure is permanent. As with LHRH agonists, side effects can be significant. They include: Loss of sex drive, hot flashes, development of breasts (gynecomastia) or painful breasts, loss of muscle, weight gain, fatigue, and decrease in levels of “good” cholesterol.

Hormone therapy for prostate cancer can cause osteoporosis, which can lead to broken bones. However, treatment with bisphosphonates — like ArediaFosamax, and Zometa — may help prevent this condition from developing.

Hormone (androgen deprivation) therapy for prostate cancer

http://www.cancer.org/cancer/prostatecancer/detailedguide/prostate-cancer-treating-hormone-therapy

Hormone therapy is also called androgen deprivation therapy (ADT) or androgen suppression therapy. The goal is to reduce levels of male hormones, called androgens, in the body, or to stop them from affecting prostate cancer cells.

The main androgens are testosterone and dihydrotestosterone (DHT). Most of the body’s androgens come from the testicles, but the adrenal glands also make a small amount. Androgens stimulate prostate cancer cells to grow. Lowering androgen levels or stopping them from getting into prostate cancer cells often makes prostate cancers shrink or grow more slowly for a time. But hormone therapy alone does not cure prostate cancer.

Hormone therapy may be used:

  • If the cancer has spread too far to be cured by surgery or radiation, or if you can’t have these treatments for some other reason
  • If your cancer remains or comes back after treatment with surgery or radiation therapy
  • Along with radiation therapy as initial treatment if you are at higher risk of the cancer coming back after treatment (based on a high Gleason score, high PSA level, and/or growth of the cancer outside the prostate)
  • Before radiation to try to shrink the cancer to make treatment more effective

Several types of hormone therapy can be used to treat prostate cancer. Some lower the levels of testosterone or other androgens (male hormones). Others block the action of those hormones.

Luteinizing hormone-releasing hormone (LHRH) analogs

These drugs lower the amount of testosterone made by the testicles. Treatment with these drugs is sometimes calledchemical castration or medical castration because they lower androgen levels just as well as orchiectomy.

Even though LHRH analogs (also called LHRH agonists or GnRH agonists) cost more than orchiectomy and require more frequent doctor visits, most men choose this method. These drugs allow the testicles to remain in place, but the testicles will shrink over time, and they may even become too small to feel.

LHRH analogs are injected or placed as small implants under the skin. Depending on the drug used, they are given anywhere from once a month up to once a year. The LHRH analogs available in the United States include leuprolide (Lupron®, Eligard®), goserelin (Zoladex®), triptorelin (Trelstar®), and histrelin (Vantas®).

When LHRH analogs are first given, testosterone levels go up briefly before falling to very low levels. This effect is called flare and results from the complex way in which LHRH analogs work. Men whose cancer has spread to the bones may have bone pain. If the cancer has spread to the spine, even a short-term increase in tumor growth as a result of the flare could compress the spinal cord and cause pain or paralysis. Flare can be avoided by giving drugs called anti-androgens for a few weeks when starting treatment with LHRH analogs. (Anti-androgens are discussed further on.)

Degarelix (Firmagon®)

Degarelix is an LHRH antagonist. LHRH antagonists work like LHRH agonists, but they reduce testosterone levels more quickly and do not cause tumor flare like the LHRH agonists do.

This drug is used to treat advanced prostate cancer. It is given as a monthly injection under the skin. The most common side effects are problems at the injection site (pain, redness, and swelling) and increased levels of liver enzymes on lab tests. Other side effects are discussed in detail below.

Abiraterone (Zytiga®)

Drugs such as LHRH agonists can stop the testicles from making androgens, but other cells in the body, including prostate cancer cells themselves, can still make small amounts, which can fuel cancer growth. Abiraterone blocks an enzyme called CYP17, which helps stop these cells from making androgens.

Abiraterone can be used in men with advanced castrate-resistant prostate cancer (cancer that is still growing despite low testosterone levels from an LHRH agonist, LHRH antagonist, or orchiectomy). Abiraterone has been shown to shrink or slow the growth of some of these tumors and help some of these men live longer.

This drug is taken as pills every day. This drug doesn’t stop the testicles from making testosterone, so men who haven’t had an orchiectomy need to continue treatment with an LHRH agonist or antagonist. Because abiraterone also lowers the level of some other hormones in the body, prednisone (a cortisone-like drug) needs to be taken during treatment as well to avoid certain side effects.

Drugs that stop androgens from working

Anti-androgens

Androgens have to bind to a protein in the cell called an androgen receptor to work. Anti-androgens are drugs that bind to these receptors so the androgens can’t.

Drugs of this type, such as flutamide (Eulexin®), bicalutamide (Casodex®), and nilutamide (Nilandron®), are pills taken daily.

Anti-androgens are not often used by themselves in the United States. An anti-androgen may be added to treatment if orchiectomy, an LHRH analog, or LHRH antagonist is no longer working by itself. An anti-androgen is also sometimes given for a few weeks when an LHRH analog is first started to prevent a tumor flare.

Anti-androgen treatment can be combined with orchiectomy or an LHRH analog as first-line hormone therapy. This is called combined androgen blockade (CAB).

9.6.2 Diabetes and Cardiovascular Disease During Androgen Deprivation Therapy for Prostate Cancer

Nancy L. KeatingA. James O’Malley and Matthew R. Smith
JCO Sep 20, 2006; 24(27):4448-4456
http://dx.doi.org:/10.1200/JCO.2006.06.2497

Purpose Androgen deprivation therapy with a gonadotropin-releasing hormone (GnRH) agonist is associated with increased fat mass and insulin resistance in men with prostate cancer, but the risk of obesity-related disease during treatment has not been well studied. We assessed whether androgen deprivation therapy is associated with an increased incidence of diabetes and cardiovascular disease. Patients and Methods Observational study of a population-based cohort of 73,196 fee-for-service Medicare enrollees age 66 years or older who were diagnosed with locoregional prostate cancer during 1992 to 1999 and observed through 2001. We used Cox proportional hazards models to assess whether treatment with GnRH agonists or orchiectomy was associated with diabetes, coronary heart disease, myocardial infarction, and sudden cardiac death. Results More than one third of men received a GnRH agonist during follow-up. GnRH agonist use was associated with increased risk of incident diabetes (adjusted hazard ratio [HR], 1.44; P < .001), coronary heart disease (adjusted HR, 1.16; P < .001), myocardial infarction (adjusted HR, 1.11; P = .03), and sudden cardiac death (adjusted HR, 1.16; P = .004). Men treated with orchiectomy were more likely to develop diabetes (adjusted HR, 1.34; P < .001) but not coronary heart disease, myocardial infarction, or sudden cardiac death (all P > .20). Conclusion GnRH agonist treatment for men with locoregional prostate cancer may be associated with an increased risk of incident diabetes and cardiovascular disease.

9.6.3 Breast Cancer and Hormone Therapy

http://www.webmd.com/breast-cancer/hormone-therapy-overview

There are certain hormones that can attach to breast cancer cells and affect their ability to multiply. The purpose of hormone therapy, also called endocrine therapy, is to add, block, or remove hormones.

With breast cancer, the female hormones estrogen andprogesterone can promote the growth of some breast cancer cells. Therefore in some patients, hormone therapy is given to block the body’s naturally occurring estrogen to slow or stop the cancer‘s growth.

There are two types of hormone therapy for breast cancer.

  • Drugs that inhibit estrogen and progesterone from promotingbreast cancer cell growth.
  • Drugs or surgery to turn off the production of hormones from the ovaries.

Faslodex, a estrogen receptor antagonist, binds to estrogen receptors and blocks their effects on cancer cells. Given as an injection, the drug is for HER2-positive metastatic disease in postmenopausal women who have already tried anti-estrogen therapy. Common side effects of Faslodex include:

  • Injection site pain
  • Nausea and vomiting
  • Loss of appetite
  • Weakness, fatigue
  • Hot flashes
  • Cough
  • Muscle, joint, and bone pain
  • Constipation
  • Shortness of breath

Zoladex and Lupron for Breast Cancer

Zoladex and Lupron are drugs that stop the production of estrogen by the ovaries. They are used in premenopausal women for the treatment of estrogen sensitive breast cancer.

Side effects of Zoladex and Lupron include:

  • Fluid retention
  • Hot flashes
  • Irregular menstrual periods
  • Pain at the injection site

http://www.cancer.gov/types/breast/breast-hormone-therapy-fact-sheet

Hormone-sensitive breast cancer cells contain proteins known as hormone receptors that become activated when hormones bind to them. The activated receptors cause changes in the expression of specific genes, which can lead to the stimulation of cell growth.

To determine whether breast cancer cells contain hormone receptors, doctors test samples of tumor tissue that have been removed by surgery. If the tumor cells contain estrogen receptors, the cancer is called estrogen receptor-positive (ER-positive), estrogen-sensitive, or estrogen-responsive. Similarly, if the tumor cells contain progesterone receptors, the cancer is called progesterone receptor-positive (PR- or PgR-positive). Approximately 70 percent of breast cancers are ER-positive. Most ER-positive breast cancers are also PR-positive (1).

Breast cancers that lack estrogen receptors are called estrogen receptor-negative (ER-negative). These tumors are estrogen-insensitive, meaning that they do not use estrogen to grow. Breast tumors that lack progesterone receptors are called progesterone receptor-negative (PR- or PgR-negative).

Hormone therapy (also called hormonal therapy, hormone treatment, or endocrine therapy) slows or stops the growth of hormone-sensitive tumors by blocking the body’s ability to produce hormones or by interfering with hormone action. Tumors that are hormone-insensitive do not respond to hormone therapy.

Hormone therapy for breast cancer is not the same as menopausal hormone therapy or female hormone replacement therapy, in which hormones are given to reduce the symptoms of menopause.

Several strategies have been developed to treat hormone-sensitive breast cancer, including the following:

Blocking ovarian function: Because the ovaries are the main source of estrogen in premenopausal women, estrogen levels in these women can be reduced by eliminating or suppressing ovarian function. Blocking ovarian function is called ovarian ablation.

Ovarian ablation can be done surgically in an operation to remove the ovaries (called oophorectomy) or by treatment with radiation. This type of ovarian ablation is usually permanent.

Alternatively, ovarian function can be suppressed temporarily by treatment with drugs called gonadotropin-releasing hormone (GnRH) agonists, which are also known as luteinizing hormone-releasing hormone (LH-RH) agonists. These medicines interfere with signals from the pituitary gland that stimulate the ovaries to produce estrogen.

Examples of ovarian suppression drugs that have been approved by the U.S. Food and Drug Administration (FDA) are goserelin (Zoladex®) and leuprolide (Lupron®).

Blocking estrogen production: Drugs called aromatase inhibitors can be used to block the activity of an enzyme called aromatase, which the body uses to make estrogen in the ovaries and in other tissues. Aromatase inhibitors are used primarily in postmenopausal women because the ovaries in premenopausal women produce too much aromatase for the inhibitors to block effectively. However, these drugs can be used in premenopausal women if they are given together with a drug that suppresses ovarian function.

Examples of aromatase inhibitors approved by the FDA are anastrozole (Arimidex®) and letrozole (Femara®), both of which temporarily inactivate aromatase, and exemestane (Aromasin®), which permanently inactivates the enzyme.

Blocking estrogen’s effects: Several types of drugs interfere with estrogen’s ability to stimulate the growth of breast cancer cells:

  • Selective estrogen receptor modulators (SERMs) bind to estrogen receptors, preventing estrogen from binding. Examples of SERMs approved by the FDA are tamoxifen (Nolvadex®), raloxifene (Evista®), andtoremifene (Fareston®). Tamoxifen has been used for more than 30 years to treat hormone receptor-positive breast cancer.Because SERMs bind to estrogen receptors, they can potentially not only block estrogen activity (i.e., serve as estrogen antagonists) but also mimic estrogen effects (i.e., serve as estrogen agonists). Most SERMs behave as estrogen antagonists in some tissues and as estrogen agonists in other tissues. For example, tamoxifen blocks the effects of estrogen in breast tissue but acts like estrogen in the uterus and bone.
  • Other antiestrogen drugs, such as fulvestrant (Faslodex®), work in a somewhat different way to block estrogen’s effects. Like SERMs, fulvestrant attaches to the estrogen receptor and functions as an estrogen antagonist. However, unlike SERMs, fulvestrant has no estrogen agonist effects. It is a pure antiestrogen. In addition, when fulvestrant binds to the estrogen receptor, the receptor is targeted for destruction.

There are three main ways that hormone therapy is used to treat hormone-sensitive breast cancer:

Adjuvant therapy for early-stage breast cancer: Research has shown that women treated for early-stage ER-positive breast cancer benefit from receiving at least 5 years of adjuvant hormone therapy (2). Adjuvant therapy is treatment given after the main treatment (surgery, in the case of early-stage breast cancer) to increase the likelihood of a cure.

Adjuvant therapy may include radiation therapy and some combination of chemotherapy, hormone therapy, and targeted therapyTamoxifen has been approved by the FDA for adjuvant hormone treatment of premenopausal and postmenopausal women (and men) with ER-positive early-stage breast cancer, andanastrozole and letrozole have been approved for this use in postmenopausal women.

A third aromatase inhibitorexemestane, is approved for adjuvant treatment of early-stage breast cancer in postmenopausal women who have received tamoxifen previously.

Until recently, most women who received adjuvant hormone therapy to reduce the chance of a breast cancer recurrence took tamoxifen every day for 5 years. However, with the advent of newer hormone therapies, some of which have been compared with tamoxifen in clinical trials, additional approaches to hormone therapy have become common (35). For example, some women may take an aromatase inhibitor every day for 5 years, instead of tamoxifen. Other women may receive additional treatment with an aromatase inhibitor after 5 years of tamoxifen. Finally, some women may switch to an aromatase inhibitor after 2 or 3 years of tamoxifen, for a total of 5 or more years of hormone therapy.

Decisions about the type and duration of adjuvant hormone therapy must be made on an individual basis. This complicated decision-making process is best carried out by talking with an oncologist, a doctor who specializes in cancer treatment.

Treatment of metastatic breast cancer: Several types of hormone therapy are approved to treat hormone-sensitive breast cancer that is metastatic (has spread to other parts of the body).

Studies have shown that tamoxifen is effective in treating women and men with metastatic breast cancer (6).Toremifene is also approved for this use. The antiestrogen fulvestrant can be used in postmenopausal women with metastatic ER-positive breast cancer after treatment with other antiestrogens (7).

The aromatase inhibitors anastrozole and letrozole can be given to postmenopausal women as initial therapy for metastatic hormone-sensitive breast cancer (89). These two drugs, as well as the aromatase inhibitor exemestane, can also be used to treat postmenopausal women with advanced breast cancer whose disease has worsened after treatment with tamoxifen (10).

Neoadjuvant treatment of breast cancer: The use of hormone therapy to treat breast cancer before surgery (neoadjuvant therapy) has been studied in clinical trials (11). The goal of neoadjuvant therapy is to reduce the size of a breast tumor to allow breast-conserving surgery. Data from randomized controlled trials have shown that neoadjuvant hormone therapies—in particular, aromatase inhibitors—can be effective in reducing the size of breast tumors in postmenopausal women. The results in premenopausal women are less clear because only a few small trials involving relatively few premenopausal women have been conducted thus far.

No hormone therapy has yet been approved by the FDA for the neoadjuvant treatment of breast cancer.

9.6.4 Hormone Therapy and Different Ovarian Cancers

Lina Steinrud Mørch, Ellen Løkkegaard, Anne Helms Andreasen, Susanne Krüger Kjær, Øjvind Lidegaard
Am J Epidemiol. 2012; 175(12):1234-1242
http://www.medscape.com/viewarticle/766010

Postmenopausal hormone therapy use increases the risk of ovarian cancer. In the present study, the authors examined the risks of different histologic types of ovarian cancer associated with hormone therapy. Using Danish national registers, the authors identified 909,946 women who were followed from 1995–2005. The women were 50–79 years of age and had no prior hormone-sensitive cancers or bilateral oophorectomy. Hormone therapy prescription data were obtained from the National Register of Medicinal Product Statistics. The National Cancer and Pathology Register provided data on ovarian cancers, including information about tumor histology. The authors performed Poisson regression analyses that included hormone exposures and confounders as time-dependent covariates. In an average of 8.0 years of follow up, 2,681 cases of epithelial ovarian cancer were detected. Compared with never users, women taking unopposed oral estrogen therapy had increased risks of both serous tumors (incidence rate ratio (IRR) = 1.7, 95% confidence interval: 1.4, 2.2) and endometrioid tumors (IRR = 1.5, 95% confidence interval: 1.0, 2.4) but decreased risk of mucinous tumors (IRR = 0.3, 95% confidence interval: 0.1, 0.8). Similar increased risks of serous and endometrioid tumors were found with estrogen/progestin therapy, whereas no association was found with mucinous tumors. Consistent with results from recent cohort studies, the authors found that ovarian cancer risk varied according to tumor histology. The types of ovarian tumors should be given attention in future studies.

Introduction

Ovarian cancer is the most lethal of gynecologic cancers. Unfortunately, little is known about its etiology. In recent meta-analyses, investigators have concluded that women taking postmenopausal hormone therapy (HT) have an increased risk of ovarian cancer compared with never users.[1, 2] Two large prospective studies, the Million Women Study and Danish Sex Hormone Register Study, found an overall increased risk of 30%–40%.[3, 4]

Less is known about the association between hormone use and the risk of different histologic subtypes of epithelial ovarian cancer. Other risk factors for ovarian cancer have been found to differ between mucinous and nonmucinous ovarian tumors, supporting the hypothesis of different etiologies.[5, 6]However, previous studies on HT and different types of ovarian tumors were mainly case-control studies, and the numbers of cases were small, especially for mucinous tumors.[1, 7–10] Most prospective cohort studies either did not examine tumor type[1, 4] or had incomplete information on histology.[11]

Recently, Danforth et al.[12] found that estrogen-only therapy (ET) was more strongly associated with the risk of endometrioid tumors than with the risk of other types of epithelial tumors in the Nurses’ Health Study (NHS). The Million Women Study found that with HT use, the highest risk was for serous tumors, whereas there was a lower risk of mucinous tumors.[3] Knowledge about the associations between HTs and subtypes of ovarian cancer will add to the understanding of how HT acts as a promoter of ovarian cancer carcinogenesis. Moreover, if different types of ovarian tumors are to be viewed as separate diseases, that fact should be considered when creating the study designs for future research. Therefore, the aim of the present study was to explore the risks of HT associated with different histologic types of ovarian cancer.

The study cohort was linked to the National Register of Medicinal Product Statistics using participants’ personal identification numbers as the key identifiers. The National Register of Medicinal Product Statistics includes information on the date of the redeemed prescriptions and the specific Anatomical Therapeutic Chemical code, dose, number of packages, defined daily doses, and route of administration (tablet, patch, gel, etc.) The specific Anatomical Therapeutic Chemical codes included in the present study have been described previously.[13]

The information on initiation of HT use (i.e., redeemed prescriptions) was updated daily for each individual during follow-up. The prescribed defined daily doses were used to determine the length of use. We included 4 months after the expiration of the prescription in all records of hormone exposure to account for any delay in recorded diagnoses in Danish registers, prolonged HT use for those taking less than the defined daily dose prescribed, and minor latency time. Thus, gaps between prescriptions of less than 4 month were filled prospectively; that is, a woman was classified as user of the drug at a given point in time if the dispensed supply from the last redemption had not run out or if it had run out within the last m days (where m is the allowed gap length).[14]

Because HT is likely to act as a promoter of ovarian cancer carcinogenesis with a yet unknown latency time, women currently taking hormones were categorized by the regimen that they took for the longest period during the study period. These variables were time varying; that is, if a woman began a new HT regimen, she would be recategorized if and when the time taking that regimen exceeded the amount of time she took the prior categorization HT regimen. The length of use was calculated as the time spent taking all systemic treatments during the study period. Whether a woman had taken hormones before 50 years of age but within the 11-year study period was accounted for in the hormone status categories, and the amount of time for which she took the hormones was accounted for in the duration of use category. The HT categories were HT use (never, past, current nonvaginal HT use, or other current use (i.e., current use of vaginal ET or a hormone intrauterine device)); hormone formulation (ET, estrogen/progestin therapy (EPT), or other (i.e., tibolone, raloxifene, progestin only, or vaginal estrogen)); hormone regimen (cyclic EPT, continuous EPT, or other); route of administration (oral ET, oral EPT or tibolone, dermal ET, dermal EPT, or other); duration of HT in years (never, current, 0.01–4 years, 4.01–7 years, or >7 years or use of vaginal ET or a hormone intrauterine device); and time since last use among former users (never, current, 0.01–2 years, 2.01–4 years, 4.01–6 years, or >6 years or use of vaginal ET or a hormone intrauterine device).

Ovarian Cancer Cases

Until December 31, 2002, we used the Danish Cancer Register to identify cases of primary invasive ovarian cancers and their histologies, using the International Classification of Diseases for Oncologytopography code 183.0 and morphology codes ending with a 3. At time of the present study, information from January 2003 had not been updated in the Danish Cancer Register. Thus, from 2003 onward, the Pathology Register was used for case findings and information on histology. The invasive epithelial tumors were classified as serous (codes M84413, M84603, M84613, and M90143), endometrioid (codes M83803 and M83813), mucinous (codes M84703, M84803, and M90153), clear-cell (codes M83103 and M83133), adenocarcinoma not otherwise specified (code M81403), or epithelial not otherwise specified (codes M80203, M80703, M81303, M85603, M89333, M89803, and M90003). Nonepithelial invasive tumors and borderline tumors were not included. Eight women for whom we did not have histologic information were excluded. Information on the stages of disease was available from the Danish Cancer Register until December 31, 2002.

From 1995 to 2005, a total of 909,946 perimenopausal and postmenopausal women with no previous cancer or removal of ovaries accumulated 7.3 million person-years of observation, corresponding to an average follow-up period of 8.0 years. The number of incident malignant epithelial ovarian cancers during the study period was 2,681. Of these, 1,336 were serous tumors, 377 were endometrioid tumors, 293 were mucinous tumors, 159 were clear-cell tumors, 115 were nonspecified epithelial tumors, and 401 were adenocarcinomas not otherwise specified. At the end of follow up, 63% of the women remained never users of HT, 22% were previous users, and 9% were current users. Compared with never users, hormone users were more likely to have undergone a hysterectomy (18.0% versus 6.2%) or unilateral salpingo-oophorectomy (5.7% versus 1.9%), to have been sterilized (8.4% versus 5.4%), and to be parous (80.8% versus 75.2%). The characteristics of the study population have been published previously.[4]

Compared with never users, current users of hormones had an increased risk of serous tumors (incidence rate ratio (IRR) = 1.7, 95% confidence interval (CI): 1.5, 1.9) and of endometrioid tumors (IRR = 1.7, 95% CI: 1.3, 2.2). Current use of hormones was not associated with the risk of mucinous or clear-cell tumors (Figure 1). The incidence rate ratios for serous ovarian cancer increased with duration of hormone use (0.01–4 years, IRR = 1.5, 95% CI: 1.3, 1.8; 4.01–7 years, IRR = 1.7, 95% CI: 1.4, 2.1; and >7 years, IRR = 2.1, 95% CI: 1.6, 2.8). The incidence rate ratios for other types of epithelial ovarian cancer were not consistently associated with duration of use (Figure 2).

(Enlarge Image)

Figure 1.

Incidence rate ratios of epithelial ovarian cancers associated with current use of hormone therapy, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

(Enlarge Image)

Figure 2.

Incidence rate ratios of epithelial ovarian cancers associated with durations of hormone therapy in years, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Risk estimates for clear-cell cancer are not shown because there were few cases. Bars, 95% confidence interval.

Time Since Hormone Use

We found increased incidence rate ratios for serous ovarian cancers for a period of up to 2 years after cessation of HT. Thereafter, the risk approached that observed in never users. For endometrioid tumors, the risk was not significantly increased after cessation of HT (Figure 3).

(Enlarge Image)

Figure 3.

Incidence rate ratios of serous and endometrioid ovarian cancers associated with time since last hormone therapy use in years, Danish Sex Hormone Register Study, 1995–2005. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

Estrogen Therapy

Compared with never users, women on unopposed ET had an increased risk of serous tumors (IRR = 1.7, 95% CI: 1.4, 2.1) and a tendency toward an increased risk of endometrioid tumors (IRR = 1.4, 95% CI: 0.9, 2.1). In contrast, the risk of mucinous tumors was decreased (IRR = 0.3, 95% CI: 0.1, 0.8). No association was found between ET and the risk of clear-cell tumors (IRR = 0.6, 95% CI: 0.2, 1.5) (Figure 4).

(Enlarge Image)

Figure 4.

Incidence rate ratios of epithelial ovarian cancers associated with hormone therapy, Danish Sex Hormone Register Study, 1995–2005. A) Estrogen-only therapy; B) estrogen/progestin therapy. Values were adjusted for age, period of use, number of births, hysterectomy, sterilization, unilateral oophorectomy or salpingo-oophorectomy, endometriosis, infertility, and educational level. The reference group was never users of hormone therapy (dashed line). Bars, 95% confidence interval.

Women on oral ET had a statistically significantly increased risk of endometrioid tumors (IRR = 1.5, 95% CI: 1.0, 2.4), and the risks for serous, mucinous, and clear-cell tumors were similar to the risks found for all ET. Because the risk associations between transdermal ET and ovarian cancers were based on a few cases, the data are not shown. Vaginal estrogen alone was associated with an increased risk of serous tumors (IRR = 1.4, 95% CI: 1.1, 1.9), whereas no associations were found with endometrioid, mucinous, or clear-cell tumors (data not shown).

Combined Therapy

Women on combined EPT had increased incidence rate ratios for serous tumors (IRR = 1.6, 95% CI: 1.4, 1.9) and endometrioid tumors (IRR = 2.0, 95% CI: 1.5, 2.6), whereas no associations were found with mucinous or clear-cell tumors (Figure 4). Similar risk associations were found among women on oral EPT. Because there were few cases, data for transdermal EPT are not shown.

Duration of HT

The incidence rate ratios for serous ovarian cancer increased with increased duration of ET and after 7 years reached an incidence rate ratio of 2.9 (95% CI: 1.9, 4.3). The risks for endometrioid ovarian cancer were similar for all durations of ET (Table 1).

Among women on cyclic EPT, the risk of endometrioid ovarian cancer was increased by 70%–140%, whereas the risk was not increased among women on continuous EPT. The risks for serous ovarian cancer were similar regardless of the duration of cyclic or continuous EPT (Table 1). Results from crude and adjusted analyses were almost identical (data not shown).

Stage of Disease

Overall, the associations between HT and risks of different ovarian tumors did not change after adjustment for the stage of disease (Table 2). Although the analyses were slightly weakened by a lower number of cases, the results roughly showed similar incidence rate ratios across the stages of disease (Table 2).

Discussion

The present large cohort study suggests that there is a differential influence of HT on different subtypes of ovarian cancer. Hormone users had an excess risk of serous and endometrioid tumors but not of mucinous and clear-cell cancers of the ovaries. Both combined EPT and unopposed ET were associated with increased risks of serous ovarian cancer. Furthermore, cyclic EPT and oral ET were associated with increased risks of endometrioid ovarian cancer. In contrast, no HT was associated with risk of clear-cell ovarian cancer, and women who had used ET had a decreased risk of mucinous ovarian cancer.

Serous Ovarian Cancer

Two large prospective cohort studies, the NHS and the Million Women Study, also found an increased risk of serous ovarian cancer among hormone users.[3, 12] In accordance with our finding, the Million Women Study reported an approximately 50% increased risk with HT.[3] The NHS supports our finding that increasing duration of ET is associated with increasing rate ratios for serous ovarian cancer.[12]

Endometrioid Ovarian Cancer

Although the Million Women Study found no association between any HT and the risk of endometrioid ovarian cancer, we found a 70% increased risk.[3] The NHS found a 50% increased risk of endometrioid tumors after 5 years of ET.[12] In our study, women on oral ET had an up to 2-fold increased risk of endometrioid tumors. Because ET increases the risk of endometrial cancer[15] and endometrioid ovarian tumors are histologically similar to endometrial tissue (16), it seems likely that ET acts through similar biologic mechanisms in the development of endometrioid ovarian cancer, a hypothesis suggested by Danforth et al..[12]

Furthermore, the present study suggests that women on cyclic EPT have an increased risk of endometrioid ovarian cancer, whereas the risk is not increased in women on continuous EPT. Only one study addressed the risk of endometrioid ovarian tumors among women on cyclic versus continuous EPT, and those investigators were not able to demonstrate an increased risk with cyclic or continuous EPT.[7] With regard to the development of endometrial cancer, the increased risk has been found to be confined to women on cyclic EPT.[15] Thus, it is possible that cyclic EPT acts through similar biologic mechanisms in the development of endometrioid ovarian cancer.

Mucinous Ovarian Cancer

Compared with women who were never prescribed HT, women on ET had a 70% decreased risk of mucinous ovarian cancer. The Million Women Study also found a decreased risk of approximately 30% with the use of HT.[3] A few other studies have also suggested that HT is associated with a decreased risk of mucinous ovarian cancer.[12, 17, 18] One group of mucinous tumors is similar to endocervical epithelium and another is similar to colonic epithelium.[16] Both HT in general and ET specifically have been found to decrease the risk of colon cancer.[19, 20] It therefore seems plausible that ET could also decrease the risk of mucinous ovarian cancer. Risch et al.[5] were the first to suggest different etiologies for mucinous and nonmucinous ovarian cancers, and a recent Danish study supported this hypothesis by suggesting significant differences in the risk between mucinous and nonmucinous tumors.[6]

Implications

Using the same data as in current study, Mørch et al.[4] found a 40% increase in the overall risk of ovarian cancer in current users of hormones, regardless of the duration and type of HT. However, in the present study, the risk of serous ovarian tumors increased with increasing durations of hormone use. This association was more pronounced among women using ET. After 7 years, the risk of serous ovarian cancer had increased 3-fold among women using ET compared with never users. On the other hand, restricting the analysis to mucinous tumors showed a decreased risk among women using ET. Thus, important information about a differential impact of HT, HT types, and associations with duration of hormone use are not described when different ovarian tumors are examined as a combined outcome.

Moreover, the clarification of the different associations between HT and subtypes of ovarian cancer adds to the understanding of how HT acts as a promoter of ovarian cancer carcinogenesis, as the results are in line with the current knowledge about HT-associated risks of cancers with similar epithelial origins. Because of this, it seems plausible that there is a causal association between HT and ovarian cancer. Other risk factors for ovarian cancer differ based on the type of tumor (mucinous vs. nonmucinous), supporting the hypothesis of different etiologies.[5, 6] The differences should be considered in research study design and suggest that different types of ovarian tumors should be viewed as separate diseases.

Strengths of Study

To our knowledge, our nationwide cohort study is the largest conducted thus far to explore the influence of HT on the risk of histologic subtypes of epithelial ovarian cancer. The validity of our outcome is considered to be high, as data from the Cancer Register validated the diagnoses (21–23). The agreement of histologic ovarian cancer diagnoses between the Pathology Register and the Cancer Register is high, and our estimates did not depend on the source of diagnoses.[24] The information on prescribed HT is transferred electronically from all Danish pharmacies by using bar codes, eliminating recall bias. Our information on both exposures and confounders was updated daily through the national registers, making it possible for us to account for changes in exposures. We excluded women with previous cancer because it might affect both the use of hormones and the subsequent risk of ovarian cancer. Our results were adjusted for age, time period, educational level, number of births, and history of hysterectomy, sterilization, unilateral oophorectomy, salpingo-oophorectomy, endometriosis, or infertility. There was, however, no significant confounding by any of the included variables. We found no evidence of earlier detection (surveillance bias) of ovarian tumors among women on HT. Finally, the stage of disease did not bias the differential association between HT and different tumor types.

Limitations of Study

Data from the National Register of Medicinal Product Statistics is not complete for the time period before January 1995. Thus, information about prescriptions for oral contraceptive use was not available for the women in current study who were 50 years of age or older from 1995−2005. Our incidence rate ratios may be underestimated because of confounding by use of oral contraceptives, as oral contraceptive use decreases the risk of ovarian cancer and often leads to HT.[25, 26] We were not able to restrict our analyses to nonobese women. The ovarian cancer risk associated with HT use is probably clearer in nonobese women (i.e., in women with a body mass index, measured as weight in kilograms divided by height in meters squared, <30).[27] Consequently, our results might be underestimated among nonobese. However, the Million Women Study adjusted data for oral contraceptive use, body mass index, age at menopause, alcohol consumption, smoking, and physical activity, and the adjustments did not result in material changes in their estimates.[3] Also, the NHS reported only minimal changes in the association between HT and the risk of ovarian cancer after adjustment for relevant potential confounders, including duration of oral contraceptive use, occurrence of natural menopause, and age at menarche.[12] The lack of information on family history of cancer might have caused an underestimation of risk in our results, as women with a family history of cancer are probably less likely to use hormones. Information on women who underwent surgical procedures was not available in the registers for the oldest women. Hysterectomy and oophorectomy reduce the risk of ovarian cancer and often lead to HT use, probably causing an underestimation of risk in older women in our results. However, despite our uneven adjustment for confounders, the risks for ovarian tumors were nearly identical across age groups.

9.6.5 Chemotherapy versus hormonal treatment in platinum- and paclitaxel-refractory ovarian cancer: a randomised trial of the German Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) Study Group Ovarian Cancer

  1. du Bois, W. Meier, H. J. Lück, G. Emons, V. Moebus, et al.
    Ann Oncol (2002) 13 (2): 251-257
    http://dx.doi.org://10.1093/annonc/mdf038

The majority of patients with ovarian cancer are not cured by first-line treatment.Until now, no study could demonstrate any substantial benefit when exposing ovarian cancer patients to second-line chemotherapy. However, most treatment regimens induce toxicity, thus negatively influencing the quality of rather limited life spans. Here we evaluate whether a second-line chemotherapy can offer any benefit compared with a less toxic hormonal treatment. Patients and methods Patients with ovarian cancer progressing during platinum-paclitaxel containing first-line therapy or experiencing relapse within 6 months were eligible. Patients were stratified for response to primary treatment (progression versus no change/response), and measurable versus non-measurable disease. Treatment consisted of either treosulfan 7 g/m2infused over 30 min or leuprorelin 3.75 mg injected subcutaneously or intramuscularly. Both regimens were repeated every 4 weeks. Results This study began in late 1996, and after 2.5 years accrual an interim analysis was performed when several investigators reported their concern about a suspected lack of efficacy. Following this analysis the recruitment was stopped early and the 78 patients already enrolled were followed up. The majority of patients received treatment until progressive disease was diagnosed or death occurred. Treatment delay was observed rarely and dose reduction was performed only in the treosulfan arm in 5% of 150 courses. Overall, both treatment arms were well tolerated. No objective responses were observed. The median survival time was 36 and 30 weeks in the treosulfan and leuprorelin arms, respectively. Overall survival did not differ between patients with relapse 3–6 months after first-line chemotherapy compared with patients with progressive disease within 3 months.

Conclusions The selected patient population represents a subgroup with extremely poor prognosis. Accordingly, results were not impressive. Both treatment arms showed favourable toxicity data, but failed to show remarkable activity, thus adding only limited evidence to the issue of whether patients with refractory ovarian cancer might benefit from second-line chemotherapy. Even stratified analysis did not identify any subgroup of patients in whom the administration of second-line chemotherapy could demonstrate a clinically relevant survival benefit.

Despite the considerable progress that has been achieved in the treatment of advanced ovarian cancer during the last de-cades, the majority of patients are still not cured by first-line treatment. Therefore, development of effective second-line treatment strategies remains a clinically relevant issue. Today standard first-line regimens in many countries contain both paclitaxel and a platinum analogue (e.g. cisplatin [12] or carboplatin [35]). There are only limited data available reporting results gained from second-line therapy following failure of this new first-line regimen. Until now, no guidelines for the selection of second-line treatment regimens have reached universal acceptance [6]. Furthermore, the definitions of recurrent or relapsed disease according to the status of platinum resistance [7] were solely based on data from patients who did not receive the actual standard first-line regimens containing paclitaxel, and therefore have to be re-evaluated. The treatment-free interval, which offers a chance of gaining a benefit from re-treatment with paclitaxel and/or platinum, remains to be defined. However, patients progressing during or relapsing shortly after platinum-paclitaxel probably have a poor prognosis and can be regarded as refractory to both of the drugs they were exposed to. Until now, no study has demonstrated clearly any substantial benefit for these patients when treating them with second-line chemotherapy. However, most treatment regimens induce toxicity, thus negatively influencing the quality of rather limited life spans in this strictly palliative setting. Therefore, the AGO Study Group set about evaluating whether a second-line chemotherapy could offer any benefit compared with a less toxic hormonal treatment.

The decision to use an alkylating agent for second-line chemotherapy was based on the assumption that these agents, which had been part of first-line treatment of ovarian cancer for decades, could offer some benefit as second-line agent after removal from first-line regimens. Treosulfan (Ovastat®, medac, Germany) was chosen as alkylating agent because it has been registered and used frequently in older first-line regimens in Germany, due to a more favourable non-haematological toxicity profile compared with cyclophosphamide [89]. The published data for treosulfan as second-line treatment after platinum failure had been partially contradictory. Two studies using intravenous treosulfan reported response rates of up to 20% in 25 and 72 patients, respectively [1011]. The latter trial included 43 patients with platinum refractory ovarian cancer and showed a 21% response rate. Orally administered treosulfan resulted in response rates of 3, 14 and 19% in 30, 22 and 16 platinum pre-treated patients, respectively [1214]. The only study reporting results of oral treosulfan in platinum refractory patients observed only one response in 30 patients. Therefore, we decided to use intravenous treosulfan as standard chemotherapy arm in this trial.

Leuprorelin (leuproreline acetate; Enantone®, Takeda, Germany), a gonadotropin-releasing hormone (GnRH) analogue, was selected as hormonal treatment in the experimental arm of this study. It could be administered in a similar time schedule as the chemotherapy regimen (monthly injections) and had shown some activity in previously reported studies in platinum pre-treated ovarian cancer. In these trials, leuprorelin had been used either as single agent [1517] or in combination with megestrole acetate or tamoxifen [1819]. Overall, nine responses have been reported in 46 platinum pre-treated patients [cumulative odds ratio (OR) 19.6%; 95% confidence interval (CI) 9% to 34%]. A retrospective review reported higher efficacy for leuprorelin compared with goserelin, thus providing further support for selecting leuprorelin in favour of other GnRH analogues [17]. However, platinum resistance had been reported inconsistently in all these studies, thus leaving some questions unanswered regarding efficiency in this particular group of patients. Toxicity profiles of leuprorelin had been uniformely reported as being mild, making this option potentially useful in this strictly palliative setting. Tamoxifen, another hormonal treatment with an 11% overall response rate reported in a meta-analysis in recurrent ovarian cancer [20], was not selected for this study, because the study group felt that the different mode of application could hamper comparability.

The median observation period was 22.5 months for all patients. The early termination of recruitment resulted in a statistical power of 80% to detect a 20% survival difference (50% versus 69.9%) after 6 months with two-sided testing and an α error of 0.05.

Treatment and tolerability

The majority of patients received treatment until progressive disease was diagnosed or death occurred. The mean and median treatment periods, respectively, were 18 and 16 weeks in the treosulfan arm, and 13 and 10 weeks in the leuprorelin arm. Treatment delay was observed rarely and median intervals per course were 30.8 and 28.6 days in the treosulfan and leuprorelin arms, respectively. Dose reduction was performed only in the treosulfan arm in eight of 150 courses (5%) because of myelosuppression.

Overall, 150 chemotherapy courses and 122 hormonal treatment courses were evaluable for toxicity. Haematological toxicities higher than grade 2 were observed in only a few patients. Thrombocytopenia grade 3/4 occurred in four and one courses in the treosulfan and leuprorelin arms, respectively. Neutropenia grade 3/4 was only observed in one course in each arm and no infections or neutropenic fever was reported. Anaemia greater than grade 2 was observed after seven courses in the treosulfan arm and after two courses in the leuprorelin arm.

Non-haematological toxicities grade 3 or 4 were reported in only few patients in both arms. Treosulfan induced nausea and emesis in 9% of patients compared with 6% of patients in the leuprorelin arm. Hot flushes were reported by one patient in each arm. Three further patients in the treosulfan arm reported grade 3 pain (two patients) and neurotoxicity (one). The latter was due to remaining toxicity from prior platinum-paclitaxel. Alopecia was common but was due to prior treatment. Re-growth of patients’ hair took longer in the treosulfan arm than in the leuprorelin arm. About one-third of patients still had alopecia after treatment with treosulfan compared with 11% in the leuprorelin arm. Fatigue was reported more frequently in the chemotherapy arm (eight of 36 patients versus one of 37 patients, treosulfan versus leuprorelin;P = 0.014, Fisher’s exact test). Overall, both treatment arms were relatively well tolerated resulting in only one treatment cessation due to toxicity.

Efficacy

No objective responses were observed in either of the treatment arms. Disease stabilisation lasting at least 4 weeks (no change) was reported in nine and four patients in the chemotherapy and hormonal treatment arm, respectively. All but one patient showed progressive disease within a median observation period of 22 months. Median progression-free survival was 17 weeks for treosulfan and 10 weeks for leuprorelin (P = 0.035, Wilcoxon test). The difference between both treatment arms remained significant in favour of treosulfan after adjusting for treatment-free interval before study entry (P = 0.028). However, after 6 months only 23% and 14% of patients in the treosulfan and leuprorelin arms had not progressed; corresponding figures for the 12 month observation period were 9% and 5%, respectively (Figure 1).

View larger version:

Figure 1. Progression-free survival (median 17 and 10 weeks for treosulfan and leuprorelin, respectively; P<0.05, log rank test; Kaplan–Meier curves).

At the time of this analysis, seven patients in the treosulfan arm and eight patients in the leuprorelin arm are still alive with disease [hazard ratio (HR) 0.98; 95% CI 0.58–1.67]. The differences observed between the treatment arms did not reach statistical significance (P = 0.87, Wilcoxon test; Figure2). Again, adjusting for a treatment-free interval before study entry did not alter results. The median survival time was 36 and 30 weeks in the treosulfan and leuprorelin arm, respectively. About one-third of patients in each arm were alive after 12 months.

View larger version:

Figure 2. Survival (median 36 and 30 weeks for treosulfan and leuprorelin, respectively; P = 0.87, log rank test; Kaplan–Meier curves).

The impact of time to treatment failure after first-line chemo-therapy on second-line therapy efficacy was analysed bi-categorially. The cut-off was set at 13 weeks, thus comparing patients with progression under first-line therapy or early relapse within 3 months with patients who relapsed 3–6 months after completion of first-line chemotherapy. Overall, the difference between the groups with respect to progression-free survival did not reach statistical significance. Median progression-free survival was 11 and 12 weeks, respectively, for the two groups (P = 0.46, log rank test; HR 0.83; 95% CI 0.51–1.35). Furthermore, overall survival did not differ significantly between patients with relapse 3–6 months after first-line chemotherapy compared with patients with progressive disease within 3 months (P = 0.34, log rank test; HR 0.77; 95% CI 0.46–1.31). However, median survival was slightly superior in the group with a longer progression-free interval after first-line therapy (42 versus 25 weeks). The latter difference did not reach statistical significance and the Kaplan–Meier curves almost fell on top of each other shortly after 1 year of observation (data not shown).

The presence of bi-dimensionally measurable disease had a negative impact on treatment results. Patients with measurable disease showed a median progression-free survival of 11 weeks compared with 19 weeks in patients with non-measurable disease (P = 0.0006, log rank test). Again, overall survival was superior in the group of patients with non-measurable disease, but this difference did not reach statistical significance (median 47 versus 24 weeks; P = 0.18, log rank test). Only 29% of patients with measurable disease compared with 46% of patients with non-measurable disease were alive after 12 months (HR 1.93; 95% CI 0.73–5.16).

Subsequent treatment

In the treosulfan arm, 15 patients received third-line treatments, of whom three were changed over to leuprorelin. The remaining eight patients received: radiotherapy (one), tamoxifen (one) or chemotherapeutic drugs [topotecan (six), etoposide (one), liposomal doxorubicin (one), carboplatin (one), carboplatin-paclitaxel (one)]. Furthermore, 14 patients received fourth-line treatment, including tamoxifen (two), MPA (one), etoposide (two), topotecan (two), and one patient each idarubicin, gemcitabin or mitoxantrone i.p. Finally,three patients received fifth-line cyclophosphamide (one), etoposide (one) or radiotherapy (one). In the leuproreline arm, almost all patients received third-line therapy. Sixteen patients were crossed over to treosulfan, four received intraperitoneal mitoxantrone, two had liposomal doxorubicin and one patient each received etoposide, topotecan, carboplatin, paclitaxel-mitoxantrone or carboplatin-paclitaxel. Two patients received hormonal third-line treatment (one each received tamoxifen and MPA). Fourth-line treatment was offered to seven patients, including radiotherapy (one), topotecan (two), and one patient each liposomal doxorubicin-etoposide, etoposide or etoposide–5-fluorouracil (5-FU). Fifth-line treatment was offered to three patients, including paclitaxel, gemcitabin and 5-FU–platinum. The considerable use of third-line therapies after progression of disease might have hampered survival analysis, which in fact showed no significant difference between the treatment arms (although progression-free survival differed).

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Discussion

This study represents a prospectively randomised trial in a very homogenous population. Only patients who were refractory to the standard first-line treatment of advanced ovarian cancer (i.e. platinum plus paclitaxel) were recruited. This selection represents a patient group with an extremely poor prognosis. At the moment, there is only limited evidence that these patients benefit from second-line chemotherapy at all, and more studies in this subgroup are necessary before any recommendations or guidelines can be established.

A randomised trial of the National Cancer Institute of Canada has shown an advantage for one arm over another when comparing 3-weekly topotecan days 1–5 to weekly topotecan in 78 patients, of whom 60% had received prior paclitaxel,and 60% were platinum refractory [23]. This advantage was limited to overall response (23% versus 8%). Progression-free survival differed only at a non-significant level (8 versus 13 weeks), and overall survival did not differ at all. Our trial showed a statistically significant advantage of one arm (treosulfan) with respect to progression-free survival, but failed to show any difference in overall survival. In addition, no differences with respect to response rates were observed. In fact, we did not observe any objective response. The latter could indicate a lack of activity of both study drugs, treosulfan and leuprorelin. However, even higher response rates as reported in the literature did not translate to longer progression-free and overall survival. A prospectively randomised trial comparing liposomal doxorubicin with topotecan included 254 platinum refractory patients; in addition, about two-thirds had received paclitaxel as part of prior therapy [24]. No significant differences were observed in the refractory subgroup: response rates were 7% and 12%, median progression-free survival was 9 and 14 weeks, and median survival was 33 and 37 weeks, respectively. Our observations of median progression free survival of 11 and 17 weeks and median survival of 30 and 36 weeks fit well with the reported data in this poor prognostic subgroup, although we did not observe any objective responses. Another randomised trial in 81 platinum refractory patients comparing paclitaxel with paclitaxel–epirubicin reported response rates of 17% and 34% translating to 2-year survival of 10% and 18% [25]. The corresponding 2-year survival in our trial was 19% and 22%, thus indicating the limited value of objective response rates as predictors for survival or progression-free survival in this poor prognostic subgroup of patients with truely refractory ovarian cancer.

Nevertheless, achieving an objective response might be beneficial in this palliative setting, especially if bulky tumours induce symptoms such as pain or bowl obstruction. However, objective response rates might not sufficiently reflect this potential benefit. Therefore, different response criteria that better reflect the palliative approach in these patients should be evaluated prospectively (e.g. symptom relief, reduction of pain medication or ability of enteral food intake). The development of better tools for the evaluation of genuine second-line chemotherapies becomes even more necessary when taking into account the fact that ovarian cancer becomes more of a chronic disease: mortality does not change substantially, but median and 5-year survival improves, thus indicating a growing need for efficient second-line and higher treatment. These therapies should allow tumour control and simultaneously should not reduce life quality.

This study reports mild toxicity data for both treatment arms, treosulfan and leuprorelin acetate, but, due to the very poor activity levels observed in both arms, adds only limited evidence to the issue of whether patients with refractory ovarian cancer benefit from second-line chemotherapy at all. Even stratified analysis in patients with progressive ovarian cancer versus patients experiencing relapse 3–6 months after first-line therapy, or patients with measurable versus non-measurable diseases, did not demonstrate any subgroup of patients in whom the administration of treosulfan second-line chemotherapy could demonstrate a clinically relevant benefit. Although a very short progression-free interval and the presence of bi-dimensionally measurable disease seemed to turn a bad prognosis into a worse prognosis, none of the differences between the strata showed a consistent and clinically relevant difference in survival. Only progression-free survival was influenced by these factors to some extent. Our data did not indicate that patients with a progression-free interval of >3 months but

However, results were disappointing in all subgroups. A rather small benefit was traded for a higher rate of fatigue in patients receiving chemotherapy. A gain of 6 weeks median progression-free survival in the superior chemotherapy arm in our study and some advantages with respect to response rates in other trials do not convincingly answer the question of whether second-line chemotherapy offers any benefit for patients with refractory ovarian cancer. Further studies are necessary to help to evaluate whether chemotherapy has a role in this subgroup of patients with a very unfavourable prognosis. A randomised comparison between best supportive care and the most active chemotherapy regimen available could theoretically be an appropriate design for such a trial. However, the German AGO investigators did not even broadly accept a randomisation between a hormonal treatment and a chemotherapy arm, as measured by an extremely slow recruitment rate. Furthermore, this study had to be closed prematurely after an interim analysis indicated only very limited activity in both treatment arms. A trial using best supportive care as one treatment arm would probably not be accepted either, although the above-mentioned efficacy data from chemotherapy studies do not provide more optimistic results.

Treosulfan showed an acceptable toxicity profile and at least some activity compared with leuprorelin acetate, thus allowing continuation of clinical research with this alkylating agent. Our subsequent trial in the refractory population compares treosulfan with topotecan (AGO protocol OVAR-2.3) and recruitment is much better, indicating that investigators more easily accept trials comparing two chemotherapy regimens. Quality of life evaluation was included in this protocol in an attempt to improve understanding of the nature of potential gains from second-line therapy.

Besides treosulfan and topotecan, which are further evaluated by our group, several chemotherapy agents have shown some activity in platinum- and paclitaxel-refractory ovarian cancer, and could serve as comparators in pending further trials: ifosfamide [26], hexamethylmelamine [27], gemcitabin [28] and liposomal doxorubicin [23,29]. The difficult task of recruiting large homogenous patient populations to second-line trials supports the ongoing discussions and activities in cooperative groups and networks, such as the worldwide Gynecologic Cancer InterGroup (GCIG), which is already planning and performing intergroup trials of second-line treatment of ovarian cancer.

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Pathway Specific Targeting in Anticancer Therapies

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

7.7 Pathway specific targeting in anticancer therapies

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

 

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Thangavelua, CQ Pana, …, BC Lowa, and J. Sivaramana
Proc Nat Acad Sci 2012; 109(20):7705–7710
http://dx.doi.org:/10.1073/pnas.1116573109

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA’s active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

The Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (12). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (23). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (25).

Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly, direct suppression of miR23a and miR23b (9) or activation of TGF-β (10) enhances KGA expression. Similarly, Rho GTPase that controls cytoskeleton and cell division also up-regulates KGA expression in an NF-κB–dependent manner (11). In addition, KGA is a substrate for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1, linking glutaminolysis to cell cycle progression (12). In comparison, function and regulation of LGA is not well studied, although it was recently shown to be linked to p53 pathway (1314). Although intense efforts are being made to develop a specific KGA inhibitor such as BPTES [bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] (15), its mechanism of inhibition and selectivity is not yet understood. Equally important is to understand how KGA function is regulated in normal and cancer cells so that a better treatment strategy can be considered.

The previous crystal structures of microbial (Mglu) and Escherichia coli glutaminases show a conserved catalytic domain of KGA (1617). However, detailed structural information and regulation are not available for human glutaminases especially the KGA, and this has hindered our strategies to develop inhibitors. Here we report the crystal structure of the catalytic domain of human apo KGA and its complexes with substrate (L-glutamine), product (L-glutamate), BPTES, and its derived inhibitors. Further, Raf-Mek-Erk module is identified as the regulator of KGA activity. Although BPTES is not recognized in the active site, its binding confers a drastic conformational change of a key loop (Glu312-Pro329), which is essential in stabilizing the catalytic pocket. Significantly, EGF activates KGA activity, which can be abolished by the kinase-dead, dominant negative mutants of Mek2 (Mek2-K101A) or its upstream activator Raf-1 (Raf-1-K375M), which are the kinase components of the growth-promoting Raf-Mek2-Erk signaling node. Furthermore, coexpression of phosphatase PP2A and treatment with Mek-specific inhibitor or alkaline phosphatase all abolished enhanced KGA activity inside the cells and in vitro, indicating that stimulation of KGA is phosphorylation dependent. Our results therefore provide mechanistic insights into KGA inhibition by BPTES and its regulation by EGF-mediated Raf-Mek-Erk module in cell growth and possibly cancer manifestation.

Structures of cKGA and Its Complexes with L-Glutamine and L-Glutamate.
The human KGA consists of 669 amino acids. We refer to Ile221-Leu533 as the catalytic domain of KGA (cKGA) (Fig. 1A). The crystal structures of the apo cKGA and in complex with L-glutamine or L-glutamate were determined (Table S1). The structure of cKGA has two domains with the active site located at the interface. Domain I comprises (Ile221-Pro281 and Cys424 -Leu533) of a five-stranded anti-parallel β-sheet (β2↓β1↑β5↓β4↑β3↓) surrounded by six α-helices and several loops. The domain II (Phe282-Thr423) mainly consists of seven α-helices. L-Glutamine/L-glutamate is bound in the active site cleft (Fig. 1B and Fig. S1B). Overall the active site is highly basic, and the bound ligand makes several hydrogen-bonding contacts to Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Fig. 1C and Fig. S1C), and these residues are highly conserved among KGA homologs (Fig. S1D). Notably, the putative serine-lysine catalytic dyad (286-SCVK-289), corresponding to the SXXK motif of class D β-lactamase (17), is located in close proximity to the bound ligand. In the apo structure, two water molecules were located in the active site, one of them being displaced by glutamine in the substrate complex. The substrate side chain is within hydrogen-bonding distance (2.9 Å) to the active site Ser286. Other key residues involved in catalysis, such as Lys289, Tyr414, and Tyr466, are in the vicinity of the active site. Lys289 is within hydrogen-bonding distance to Ser286 (3.1 Å) and acts as a general base for the nucleophilic attack by accepting the proton from Ser286. Tyr466, which is close to Ser286 and in hydrogen-bonding contact (3.2 Å) with glutamine, is involved in proton transfer during catalysis. Moreover, the carbonyl oxygen of the glutamine is hydrogen-bonded with the main chain amino groups of Ser286 and Val484, forming the oxyanion hole. Thus, we propose that in addition to the putative catalytic dyad (Ser286 XX Lys289), Tyr466 could play an important role in the catalysis (Fig. 1Cand Fig. S2).

structure of the cKGA-L-glutamine complex

structure of the cKGA-L-glutamine complex

http://www.pnas.org/content/109/20/7705/F1.medium.gif

Fig. 1.  Schematic view and structure of the cKGA-L-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (L-glutamine) is shown as a cyan stick. (C) Fourier 2Fo-Fc electron density map (contoured at 1 σ) for L-glutamine, that makes hydrogen bonds with active site residues are shown.

Allosteric Binding Pocket for BPTES. The chemical structure of BPTES has an internal symmetry, with two exactly equivalent parts including a thiadiazole, amide, and a phenyl group (Fig. S3A), and it equally interacts with each monomer. The thiadiazole group and the aliphatic linker are well buried in a hydrophobic cluster that consists of Leu321, Phe322, Leu323, and Tyr394 from both monomers, which forms the allosteric pocket (Fig. 2 B–E). The side chain of Phe322 is found at the bottom of the allosteric pocket. The phenyl-acetamido moiety of BPTES is partially exposed on the loop (Asn324-Glu325), where it interacts with Phe318, Asn324, and the aliphatic part of the Glu325 side chain. On the basis of our observations we synthesized a series of BPTES-derived inhibitors (compounds2–5) (Fig. S3 AF and SI Results) and solved their cocrystal structure of compounds 2–4. Similar to BPTES, compounds 24 all resides within the hydrophobic cluster of the allosteric pocket (Fig. S3 CF).

Fig. 2. Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES.

Allosteric Binding of BPTES Triggers Major Conformational Change in the Key Loop Near the Active Site.  The overall structure of these inhibitor complexes superimposes well with apo cKGA. However, a major conformational change at the Glu312 to Pro329 loop was observed in the BPTES complex (Fig. 2F). The most conformational changes of the backbone atoms that moved away from the active site region are found at the center of the loop (Leu316-Lys320). The backbone of the residues Phe318 and Asn319 is moved ≈9 Å and ≈7 Å, respectively, compared with the apo structure, whereas the side chain of these residues moved ≈14 Å and ≈12 Å, respectively. This loop rearrangement in turn brings Phe318 closer to the phenyl group of the inhibitor and forms the inhibitor binding pocket, whereas in the apo structure the same loop region (Leu316-Lys320) was found to be adjacent to the active site and forms a closed conformation of the active site.

Binding of BPTES Stabilizes the Inactive Tetramers of cKGA.  To understand the role of oligomerization in KGA function, dimers and tetramers of cKGA were generated using the symmetry-related monomers (Fig. 2 A–E and Fig. S4 D and E). The dimer interface in the cKGA: BPTES complex is formed by residues from the helix Asp386-Lys398 of both monomers and involves hydrogen bonding, salt bridges, and hydrophobic interactions (Phe389, Ala390, Tyr393, and Tyr394), besides two sulfate ions located in the interface (Fig. 2E). The dimers are further stabilized by binding of BPTES, where it binds to loop residues (Glu312-Pro329) and Tyr394 from both monomers (Fig. 2 D and E). Similarly, residues from Lys311-Asn319 loop and Arg454, His461, Gln471, and Asn529-Leu533 are involved in the interface with neighboring monomers to form the tetramer in the BPTES complex.

BPTES Induces Allosteric Conformational Changes That Destabilize Catalytic Function of KGA

Fig. 3A shows that 293T cells overexpressing KGA produced higher level of glutamate compared with the vector control cells. Most significantly, all of these mutants, except Phe322Ala, greatly diminished the KGA activity.

Fig. 3. Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity.

Raf-Mek-Erk Signaling Module Regulates KGA Activity. Because KGA supports cell growth and proliferation, we first validated that treatment of cells with BPTES indeed inhibits KGA activity and cell proliferation (Fig. S5 A–D and SI Results). Next, as cells respond to various physiological stimuli to regulate their metabolism, with many of the metabolic enzymes being the primary targets of modulation (18), we examined whether KGA activity can be regulated by physiological stimuli, in particular EGF, which is important for cell growth and proliferation. Cells overexpressing KGA were made quiescent and then stimulated with EGF for various time points. Fig. 4A shows that the basal KGA activity remained unchanged 30 min after EGF stimulation, but the activity was substantially enhanced after 1 h and then gradually returned to the basal level after 4 h. Because EGF activates the Raf-Mek-Erk signaling module (19), treatment of cells with Mek-specific inhibitor U0126 could block the enhanced KGA activity with parallel inhibition of Erk phosphorylation (Fig. 4A). Interestingly, such Mek-induced KGA activity is specific to EGF and lysophosphatidic acid (LPA) but not with other growth factors, such as PDGF, TGF-β, and basic FGF (bFGF), despite activation of Mek-Erk by bFGF (Fig. S6A).

The results show that KGA could interact equally well with the wild-type or mutant forms of Raf-1 and Mek2 (Fig. 4C). Importantly, endogenous Raf-1 or Erk1/2, including the phosphorylated Erk1/2 (Fig. 4 C and D), could be detected in the KGA complex. Taken together, these results indicate that the activity of KGA is directly regulated by Raf-Mek-Erk downstream of EGF receptor. To further show that Mek2-enhanced KGA activity requires both the kinase activity of Mek2 and the core residues for KGA catalysis, wild-type or triple mutant (Leu321Ala/Phe322Ala/Leu323Ala) of KGA was coexpressed with dominant negative Mek2-KA or the constitutive active Mek2-SD and their KGA activities measured. The result shows that the presence of Mek2-KA blocks KGA activity, whereas the triple mutant still remains inert even in the presence of the constitutively active Mek2 (Fig. 4E), and despite Mek2 binding to the KGA triple mutant (Fig. S7B). Consequently, expressing triple mutant did not support cell proliferation as well as the wild-type control (Fig. S7C).

Fig. 4. EGFR-Raf-Mek-Erk signaling stimulates KGA activity.

When cells expressing both KGA and Mek2-K101A were treated with subthreshold levels of BPTES, there was a synergistic reduction in cell proliferation (Fig. S6C and SI Results). Lastly, to determine whether regulation of KGA by Raf-Mek-Erk depends on its phosphorylation status, cells were transfected with KGA with or without the protein phosphatase PP2A and assayed for the KGA activity. PP2A is a ubiquitous and conserved serine/threonine phosphatase with broad substrate specificity. The results indicate that KGA activity was reduced down to the basal level in the presence of PP2A (Fig. 5A). Coimmunoprecipitation study also revealed that KGA interacts with PP2A (Fig. 5B), suggesting a negative feedback regulation by this protein phosphatase. Furthermore, treatment of immunoprecipitated and purified KGA with calf-intestine alkaline phosphatase (CIAP) almost completely abolished the KGA activity in vitro (Fig. S6D). Taken together, these results indicate that KGA activity is regulated by Raf-Mek2, and KGA activation by EGF could be part of the EGF-stimulated Raf-Mek-Erk signaling program in controlling cell growth and proliferation (Fig. 5C).

KGA activity is regulated by phosphorylation

KGA activity is regulated by phosphorylation

http://www.pnas.org/content/109/20/7705/F5.medium.gif

Fig. 5. KGA activity is regulated by phosphorylation. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation.

Small-molecule inhibitors that target glutaminase activity in cancer cells are under development. Earlier efforts targeting glutaminase using glutamine analogs have been unsuccessful owing to their toxicities (2). BPTES has attracted much attention as a selective, nontoxic inhibitor of KGA (15), and preclinical testing of BPTES toward human cancers has just begun (20). BPTES selectively suppresses the growth of glioma cells (21) and inhibits the growth of lymphoma tumor growth in animal model studies (22). Wang et al. (11) reported a small molecule that targets glutaminase activity and oncogenic transformation. Despite extensive studies, nothing is known about the structural and molecular basis for KGA inhibitory mechanisms and how their function is regulated during normal and cancer cell metabolism. Such limited information impedes our effort in producing better generations of inhibitors for better treatment regimens.

Comparison of the complex structures with apo cKGA structure, which has well-defined electron density for the key loop, we provide the atomic view of an allosteric binding pocket for BPTES and elucidate the inhibitory mechanism of KGA by BPTES. The key residues of the loop (Glu312-Pro329) undergo major conformational changes upon binding of BPTES. In addition, structure-based mutagenesis studies suggest that this loop is essential for stabilizing the active site. Therefore, by binding in an allosteric pocket, BPTES inhibits the enzymatic activity of KGA through (i) triggering a major conformational change on the key residues that would normally be involved in stabilizing the active sites and regulating its enzymatic activity; and (ii) forming a stable inactive tetrameric KGA form. Our findings are further supported by two very recent reports on KGA isoform (GAC) (2324), although these studies lack full details owing to limitation of their electron density maps. BPTES is specific to KGA but not to LGA (15). Sequence comparison of KGA with LGA (Fig. S8A) reveals two unique residues on KGA, Phe318 and Phe322, which upon mutation to LGA counterparts, become resistant to BPTES. Thus, our study provides the molecular basis of BPTES specificity.

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Islam SS, Mokhtari RB, Noman AS, …, van der Kwast T, Yeger H, Farhat WA.
Molec Carcinogenesis mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22300

shh sonic hedgehog signaling pathway nri2151-f1

shh sonic hedgehog signaling pathway nri2151-f1

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis

Sonic_hedgehog_pathway

Sonic_hedgehog_pathway

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Gaikwad SM, Thakur B, Sakpal A, Singh RK, Ray P.
Int J Biochem Cell Biol. 2015 Apr; 61:90-102
http://dx.doi.org:/10.1016/j.biocel.2015.02.001

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

Kuboki MIto ASimizu SUmezawa K.
Biochem Biophys Res Commun. 2015 Jan 16; 456(3):810-4
http://dx.doi.org:/10.1016/j.bbrc.2014.12.024

Activation of caspase 3 and caspase-dependent apoptosis  nrmicro2071-f1

Activation of caspase 3 and caspase-dependent apoptosis nrmicro2071-f1

Highlights

  • We have prepared RelB mutants that are resistant to caspase 3-induced scission.
  • Vinblastine induced caspase 3-dependent site-specific RelB cleavage in cancer cells.
  • Cancer cells expressing cleavage-resistant RelB showed less sensitivity to vinblastine.
  • Caspase 3-induced RelB cleavage may provide positive feedback mechanism in apoptosis.

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

apoptotic-caspases-control-microglia-activation-cdd2011107f3

apoptotic-caspases-control-microglia-activation-cdd2011107f3

 

 

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

He GDhar DNakagawa HFont-Burgada JOgata HJiang Y, et al.
Cell. 2013 Oct 10; 155(2):384-96
http://dx.doi.org/10.1016%2Fj.cell.2013.09.031

Il-6 signaling in cancer cells

Il-6 signaling in cancer cells

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from pre-malignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Clonal evolution and selective pressure may cause some descendants of the initial progenitor to cross the bridge of no return and form a premalignant lesion. Cancer genome sequencing indicates that most cancers require at least five genetic changes to evolve (Wood et al., 2007). It has been difficult to isolate and propagate cancer progenitors prior to detection of tumor masses. Further, it is not clear whether cancer progenitors are the precursors for the  cancer stem cells (CSCs)isolated from cancers. An answer to these critical questions depends on identification and isolation of cancer progenitors, which may also enable definition of molecular markers and signaling pathways suitable for early detection and treatment.

Hepatocellular carcinoma (HCC), the end product of chronic liver diseases, requires several decades to evolve (El-Serag, 2011). It is the third most deadly and fifth most common cancer worldwide, and in the United States its incidence has doubled in the past two decades. Furthermore, 8% of the world’s population are chronically infected with hepatitis B or C viruses (HBV and HCV) and are at a high risk of new HCC development (El-Serag, 2011). Up to 5% of HCV patients will develop HCC in their lifetime, and the yearly HCC incidence in patients with cirrhosis is 3%–5%. These tumors may arise from premalignant lesions, ranging from dysplastic foci to dysplastic hepatocyte nodules that are often seen in damaged and cirrhotic livers and are more proliferative than the surrounding parenchyma (Hytiroglou et al., 2007). There is no effective treatment for HCC and, upon diagnosis, most patients with advanced disease have a remaining lifespan of 4–6 months. Premalignant lesions, called foci of altered hepatocytes (FAH), were described in chemically induced HCC models (Pitot, 1990), but it was questioned whether these lesions harbor tumor progenitors or result from compensatory proliferation (Sell and Leffert, 2008). The aim of this study was to determine whether HCC progenitor cells (HcPCs) exist and if so, to isolate these cells and identify some of the signaling networks that are involved in their maintenance and progression.

We now describe HcPC isolation from mice treated with the procarcinogen diethyl nitrosamine (DEN), which induces poorly differentiated HCC nodules within 8 to 9 months (Verna et al., 1996). The use of a chemical carcinogen is justified because the finding of up to 121 mutations per HCC genome suggests that carcinogens may be responsible for human HCC induction (Guichard et al., 2012). Furthermore, 20%–30% of HCC, especially in HBV-infected individuals, evolve in noncirrhotic livers (El-Serag, 2011). Nonetheless, we also isolated HcPCs fromTak1Δhep mice, which develop spontaneous HCC as a result of progressive liver damage, inflammation, and fibrosis caused by ablation of TAK1 (Inokuchi et al., 2010). Although the etiology of each model is distinct, both contain HcPCs that express marker genes and signaling pathways previously identified in human HCC stem cells (Marquardt and Thorgeirsson, 2010) long before visible tumors are detected. Furthermore, DEN-induced premalignant lesions and HcPCs exhibit autocrine IL-6 production that is critical for tumorigenic progression. Circulating IL-6 is a risk indicator in several human pathologies and is strongly correlated with adverse prognosis in HCC and cholangiocarcinoma (Porta et al., 2008Soresi et al., 2006). IL-6 produced by in-vitro-induced CSCs was suggested to be important for their maintenance (Iliopoulos et al., 2009). Little is known about the source of IL-6 in HCC.

DEN-Induced Collagenase-Resistant Aggregates of HCC Progenitors

A single intraperitoneal (i.p.) injection of DEN into 15-day-old BL/6 mice induces HCC nodules first detected 8 to 9 months later. However, hepatocytes prepared from macroscopically normal livers 3 months after DEN administration already contain cells that progress to HCC when transplanted into the permissive liver environment of MUP-uPA mice (He et al., 2010), which express urokinase plasminogen activator (uPA) from a mouse liver-specific major urinary protein (MUP) promoter and undergo chronic liver damage and compensatory proliferation (Rhim et al., 1994). HCC markers such as α fetoprotein (AFP), glypican 3 (Gpc3), and Ly6D, whose expression in mouse liver cancer was reported (Meyer et al., 2003), were upregulated in aggregates from DEN-treated livers, but not in nonaggregated hepatocytes or aggregates from control livers (Figure S1A). Using 70 μm and 40 μm sieves, we separated aggregated from nonaggregated hepatocytes (Figure 1A) and tested their tumorigenic potential by transplantation into MUP-uPA mice (Figure 1B). To facilitate transplantation, the aggregates were mechanically dispersed and suspended in Dulbecco’s modified Eagle’s medium (DMEM). Five months after intrasplenic (i.s.) injection of 104 viable cells, mice receiving cells from aggregates developed about 18 liver tumors per mouse, whereas mice receiving nonaggregated hepatocytes developed less than 1 tumor each (Figure 1B). The tumors exhibited typical trabecular HCC morphology and contained cells that abundantly express AFP (Figure S1B).

Only liver tumors were formed by the transplanted cells. Other organs, including the spleen into which the cells were injected, remained tumor free (Figure 1B), suggesting that HcPCs progress to cancer only in the proper microenvironment. Indeed, no tumors appeared after HcPC transplantation into normal BL/6 mice. But, if BL/6 mice were first treated with retrorsine (a chemical that permanently inhibits hepatocyte proliferation [Laconi et al., 1998]), intrasplenically transplanted with HcPC-containing aggregates, and challenged with CCl4 to induce liver injury and compensatory proliferation (Guo et al., 2002), HCCs readily appeared (Figure 1C). CCl4 omission prevented tumor development. Notably, MUP-uPA or CCl4-treated livers are fragile, rendering direct intrahepatic transplantation difficult. CCl4-induced liver damage, especially within a male liver, generates a microenvironment that drives HcPC proliferation and malignant progression. To examine this point, we transplanted GFP-labeled HcPC-containing aggregates into retrorsine-treated BL/6 mice and examined their ability to proliferate with or without subsequent CCl4 treatment. Indeed, the GFP+ cells formed clusters that grew in size only in CCl4-treated host livers (Figure S1E). Omission of CC14 prevented their expansion.

Because CD44 is expressed by HCC stem cells (Yang et al., 2008Zhu et al., 2010), we dispersed the aggregates and separated CD44+ from CD44 cells and transplanted both into MUP-uPA mice. Whereas as few as 103 CD44+ cells gave rise to HCCs in 100% of recipients, no tumors were detected after transplantation of CD44 cells (Figure 1E). Remarkably, 50% of recipients developed at least one HCC after receiving as few as 102 CD44+ cells.

HcPC-Containing Aggregates in Tak1Δhep Mice

We applied the same HcPC isolation protocol to Tak1Δhep mice, which develop HCC of different etiology from DEN-induced HCC. Importantly, Tak1Δhep mice develop HCC as a consequence of chronic liver injury and fibrosis without carcinogen or toxicant exposure (Inokuchi et al., 2010). Indeed, whole-tumor exome sequencing revealed that DEN-induced HCC contained about 24 mutations per 106 bases (Mb) sequenced, with B-RafV637E being the most recurrent, whereas 1.4 mutations per Mb were detected inTak1Δhep HCC’s exome (Table S1). By contrast, Tak1Δhep HCC exhibited gene copy number changes. HCC developed in 75% of MUP-uPA mice that received dispersed Tak1Δhep aggregates, but no tumors appeared in mice receiving nonaggregated Tak1Δhep or totalTak1f/f hepatocytes (Figure 2B). bile duct ligation (BDL) or feeding with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), treatments that cause cholestatic liver injuries and oval cell expansion (Dorrell et al., 2011), did increase the number of small hepatocytic cell aggregates (Figure S2A). Nonetheless, no tumors were observed 5 months after injection of such aggregates into MUP-uPA mice (Figure S2B). Thus, not all hepatocytic aggregates contain HcPCs, and HcPCs only appear under tumorigenic conditions.

The HcPC Transcriptome Is Similar to that of HCC and Oval Cells

To determine the relationship between DEN-induced HcPCs, normal hepatocytes, and fully transformed HCC cells, we analyzed the transcriptomes of aggregated and nonaggregated hepatocytes from male littermates 5 months after DEN administration, HCC epithelial cells from DEN-induced tumors, and normal hepatocytes from age- and gender-matched littermate controls. Clustering analysis distinguished the HCC samples from other samples and revealed that the aggregated hepatocyte samples did not cluster with each other but rather with nonaggregated hepatocytes derived from the same mouse (Figure S3A). 57% (583/1,020) of genes differentially expressed in aggregated relative to nonaggregated hepatocytes are also differentially expressed in HCC relative to normal hepatocytes (Figure 3B, top), a value that is highly significant (p < 7.13 × 10−243). More specifically, 85% (494/583) of these genes are overexpressed in both HCC and HcPC-containing aggregates (Figure 3B, bottom table). Thus, hepatocyte aggregates isolated 5 months after DEN injection contain cells that are related in their gene expression profile to HCC cells isolated from fully developed tumor nodules.

Figure 3 Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells

We examined which biological processes or cellular compartments were significantly overrepresented in the induced or repressed genes in both pairwise comparisons (Gene Ontology Analysis). As expected, processes and compartments that were enriched in aggregated hepatocytes relative to nonaggregated hepatocytes were almost identical to those that were enriched in HCC relative to normal hepatocytes (Figure 3C). Several human HCC markers, including AFP, Gpc3 and H19, were upregulated in aggregated hepatocytes (Figures 3D and 3E). Aggregated hepatocytes also expressed more Tetraspanin 8 (Tspan8), a cell-surface glycoprotein that complexes with integrins and is overexpressed in human carcinomas (Zöller, 2009). Another cell-surface molecule highly expressed in aggregated cells is Ly6D (Figures 3D and 3E). Immunofluorescence (IF) analysis revealed that Ly6D was undetectable in normal liver but was elevated in FAH and ubiquitously expressed in most HCC cells (Figure S3C). A fluorescent-labeled Ly6D antibody injected into HCC-bearing mice specifically stained tumor nodules (Figure S3D). Other cell-surface molecules that were upregulated in aggregated cells included syndecan 3 (Sdc3), integrin α 9 (Itga9), claudin 5 (Cldn5), and cadherin 5 (Cdh5) (Figure 3D). Aggregated hepatocytes also exhibited elevated expression of extracellular matrix proteins (TIF3 and Reln1) and a serine protease inhibitor (Spink3). Elevated expression of such proteins may explain aggregate formation. Aggregated hepatocytes also expressed progenitor cell markers, including the epithelial cell adhesion molecule (EpCAM) (Figure 3E) and Dlk1 (Figure 3D). We compared the HcPC and HCC (Figure 3A) to the transcriptome of DDC-induced oval cells (Shin et al., 2011). This analysis revealed a striking similarity between the HCC, HcPC, and the oval cell transcriptomes (Figure S3B). Despite these similarities, some genes that were upregulated in HcPC-containing aggregates and HCC were not upregulated in oval cells. Such genes may account for the tumorigenic properties of HcPC and HCC.

Figure 4  DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells

We examined the aggregates for signaling pathways and transcription factors involved in hepatocarcinogenesis. Many aggregated cells were positive for phosphorylated c-Jun and STAT3 (Figure 4A), transcription factors involved in DEN-induced hepatocarcinogenesis (Eferl et al., 2003He et al., 2010). Sox9, a transcription factor that marks hepatobiliary progenitors (Dorrell et al., 2011), was also expressed by many of the aggregated cells, which were also positive for phosphorylated c-Met (Figure 4A), a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) and is essential for liver development (Bladt et al., 1995) and hepatocarcinogenesis (Wang et al., 2001). Few of the nonaggregated hepatocytes exhibited activation of these signaling pathways. Despite different etiology, HcPC-containing aggregates from Tak1Δhep mice exhibit upregulation of many of the same markers and pathways that are upregulated in DEN-induced HcPC-containing aggregates. Flow cytometry confirmed enrichment of CD44+ cells as well as CD44+/CD90+ and CD44+/EpCAM+ double-positive cells in the HcPC-containing aggregates from either DEN-treated or Tak1Δhep livers (Figure S4B).

HcPC-Containing Aggregates Originate from Premalignant Dysplastic Lesions

FAH are dysplastic lesions occurring in rodent livers exposed to hepatic carcinogens (Su et al., 1990). Similar lesions are present in premalignant human livers (Su et al., 1997). Yet, it is still debated whether FAH correspond to premalignant lesions or are a reaction to liver injury that does not lead to cancer (Sell and Leffert, 2008). In DEN-treated males, FAH were detected as early as 3 months after DEN administration (Figure 5A), concomitant with the time at which HcPC-containing aggregates were detected. In females, FAH development was delayed. FAH contained cells positive for the same progenitor cell markers and activated signaling pathways present in HcPC-containing aggregates, including AFP, CD44, and EpCAM (Figure 5C). FAH also contained cells positive for activated STAT3, c-Jun, and PCNA (Figure 5C).

HcPCs Exhibit Autocrine IL-6 Expression Necessary for HCC Progression

In situ hybridization (ISH) and immunohistochemistry (IHC) revealed that DEN-induced FAH contained IL-6-expressing cells (Figures 6A, 6B, and S5), and freshly isolated DEN-induced aggregates contained more IL-6 messenger RNA (mRNA) than nonaggregated hepatocytes (Figure 6C). We examined several factors that control IL-6 expression and found that LIN28A and B were significantly upregulated in HcPCs and HCC (Figures 6D and 6E). LIN28-expressing cells were also detected within FAH (Figure 6F). As reported (Iliopoulos et al., 2009), knockdown of LIN28B in cultured HcPC or HCC cell lines decreased IL-6 expression (Figure 6G). LIN28 exerts its effects through downregulation of the microRNA (miRNA) Let-7 (Iliopoulos et al., 2009).

Figure 6  Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression

Figure 7  HCC Growth Depends on Autocrine IL-6 Production

The isolation and characterization of cells that can give rise to HCC only after transplantation into an appropriate host liver undergoing chronic injury demonstrates that cancer arises from progenitor cells that are yet to become fully malignant. Importantly, unlike fully malignant HCC cells, the HcPCs we isolated cannot form s.c. tumors or even liver tumors when introduced into a nondamaged liver. Liver damage induced by uPA expression or CCl4 treatment provides HcPCs with the proper cytokine and growth factor milieu needed for their proliferation. Although HcPCs produce IL-6, they may also depend on other cytokines such as TNF, which is produced by macrophages that are recruited to the damaged liver. In addition, uPA expression and CCl4 treatment may enhance HcPC growth and progression through their fibrogenic effect on hepatic stellate cells. Although HCC and other cancers have been suspected to arise from premalignant/dysplastic lesions (Hruban et al., 2007Hytiroglou et al., 2007), a direct demonstration that such lesions progress into malignant tumors has been lacking. Based on expression of common markers—EpCAM, CD44, AFP, activated STAT3, and IL-6—that are not expressed in normal hepatocytes, we postulate that HcPCs originate from FAH or dysplastic foci, which are first observed in male mice within 3 months of DEN exposure.

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

Lin R1Tao RGao XLi TZhou XGuan KLXiong YLei QY.
Mol Cell. 2013 Aug 22; 51(4):506-18
http://dx.doi.org:/10.1016/j.molcel.2013.07.002

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

Fatty acid synthesis occurs at low rates in most nondividing cells of normal tissues that primarily uptake lipids from circulation. In contrast, increased lipogenesis, especially de novo lipid synthesis, is a key characteristic of cancer cells. Many studies have demonstrated that in cancer cells, fatty acids are preferred to be derived from de novo synthesis instead of extracellular lipid supply (Medes et al., 1953Menendez and Lupu, 2007;Ookhtens et al., 1984Sabine et al., 1967). Fatty acids are key building blocks for membrane biogenesis, and glucose serves as a major carbon source for de novo fatty acid synthesis (Kuhajda, 2000McAndrew, 1986;Swinnen et al., 2006). In rapidly proliferating cells, citrate generated by the tricarboxylic acid (TCA) cycle, either from glucose by glycolysis or glutamine by anaplerosis, is preferentially exported from mitochondria to cytosol and then cleaved by ATP citrate lyase (ACLY) (Icard et al., 2012) to produce cytosolic acetyl coenzyme A (acetyl-CoA), which is the building block for de novo lipid synthesis. As such, ACLY couples energy metabolism with fatty acids synthesis and plays a critical role in supporting cell growth. The function of ACLY in cell growth is supported by the observation that inhibition of ACLY by chemical inhibitors or RNAi dramatically suppresses tumor cell proliferation and induces differentiation in vitro and in vivo (Bauer et al., 2005Hatzivassiliou et al., 2005). In addition, ACLY activity may link metabolic status to histone acetylation by providing acetyl-CoA and, therefore, gene expression (Wellen et al., 2009).

While ACLY is transcriptionally regulated by sterol regulatory element-binding protein 1 (SREBP-1) (Kim et al., 2010), ACLY activity is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Berwick et al., 2002Migita et al., 2008Pierce et al., 1982). Akt can directly phosphorylate and activate ACLY (Bauer et al., 2005Berwick et al., 2002Migita et al., 2008Potapova et al., 2000). Covalent lysine acetylation has recently been found to play a broad and critical role in the regulation of multiple metabolic enzymes (Choudhary et al., 2009Zhao et al., 2010). In this study, we demonstrate that ACLY protein is acetylated on multiple lysine residues in response to high glucose. Acetylation of ACLY blocks its ubiquitinylation and degradation, thus leading to ACLY accumulation and increased fatty acid synthesis. Our observations reveal a crosstalk between protein acetylation and ubiquitylation in the regulation of fatty acid synthesis and cell growth.

Acetylation of ACLY at Lysines 540, 546, and 554

Recent mass spectrometry-based proteomic analyses have potentially identified a large number of acetylated proteins, including ACLY (Figure S1A available online; Choudhary et al., 2009Zhao et al., 2010). We detected the acetylation level of ectopically expressed ACLY followed by western blot using pan-specific anti-acetylated lysine antibody. ACLY was indeed acetylated, and its acetylation was increased by nearly 3-fold after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases, and trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) class I and class II (Figure 1A). Experiments with endogenous ACLY also showed that TSA and NAM treatment enhanced ACLY acetylation (Figure 1B).

Figure 1  ACLY Is Acetylated at Lysines 540, 546, and 554

Ten putative acetylation sites were identified by mass spectrometry analyses (Table S1). We singly mutated each lysine to either a glutamine (Q) or an arginine (R) and found that no single mutation resulted in a significant reduction of ACLY acetylation (data not shown), indicating that ACLY may be acetylated at multiple lysine residues. Three lysine residues, K540, K546, and K554, received high scores in the acetylation proteomic screen and are evolutionarily conserved from C. elegans to mammals (Figure S1A). We generated triple Q and R mutants of K540, K546, and K554 (3KQ and 3KR) and found that both 3KQ and 3KR mutations resulted in a significant (~60%) decrease in ACLY acetylation (Figure 1C), indicating that 3K are the major acetylation sites of ACLY.  Further, we found that the acetylation of endogenous ACLY is clearly increased after treatment of cells with NAM and TSA (Figure 1D). These results demonstrate that ACLY is acetylated at K540, K546, and K554.

Glucose Promotes ACLY Acetylation to Stabilize ACLY

In mammalian cells, glucose is the main carbon source for de novo lipid synthesis. We found that ACLY levels increased with increasing glucose concentration, which also correlated with increased ACLY 3K acetylation (Figure 1E). Furthermore, to confirm whether the glucose level affects ACLY protein stability in vivo, we intraperitoneally injected glucose in BALB/c mice and found that high glucose resulted in a significant increase of ACLY protein levels (Figure 1F).

To determine whether ACLY acetylation affects its protein levels, we treated HeLa and Chang liver cells with NAM and TSA and found an increase in ACLY protein levels (Figure S1G, upper panel). ACLY mRNA levels were not significantly changed by the treatment of NAM and TSA (Figure S1G, lower panel), indicating that this upregulation of ACLY is mostly achieved at the posttranscriptional level. Indeed, ACLY protein was also accumulated in cells treated with the proteasome inhibitor MG132, indicating that ACLY stability could be regulated by the ubiquitin-proteasome pathway (Figure 1G). Blocking deacetylase activity stabilized ACLY (Figure S1H). The stabilization of ACLY induced by high glucose was associated with an increase of ACLY acetylation at K540, K546, and K554. Together, these data support a notion that high glucose induces both ACLY acetylation and protein stabilization and prompted us to ask whether acetylation directly regulates ACLY stability. We then generated ACLYWT, ACLY3KQ, and ACLY3KRstable cells after knocking down the endogenous ACLY. We found that the ACLY3KR or ACLY3KQmutant was more stable than the ACLYWT (Figures 1I and S1I). Collectively, our results suggest that glucose induces acetylation at K540, 546, and 554 to stabilize ACLY.

Acetylation Stabilizes ACLY by Inhibiting Ubiquitylation

To determine the mechanism underlying the acetylation and ACLY protein stability, we first examined ACLY ubiquitylation and found that it was actively ubiquitylated (Figure 2A). Previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). In order to identify the ubiquitylation sites, we tested the ubiquitylation levels of double mutants 540R–546R and 546–554R (Figure S2A). We found that the ubiquitylation of the 540R-546R and 546R-554R mutants is partially decreased, while mutation of K540, K546, and K554 (3KR), which changes all three putative acetylation lysine residues of ACLY to arginine residues, dramatically reduced the ACLY ubiquitylation level (Figures 2B and S2A), indicating that 3K lysines might also be the ubiquitylation target residues. Moreover, inhibition of deacetylases by NAM and TSA decreased ubiquitylation of WT but not 3KQ or 3KR mutant ACLY (Figure 2C). These results implicate an antagonizing role of the acetylation towards the ubiquitylation of ACLY at these three lysine residues.

Figure 2  Acetylation Protects ACLY from Proteasome Degradation by Inhibiting Ubiquitylation

We found that ACLY acetylation was only detected in the nonubiquitylated, but not the ubiquitylated (high-molecular-weight), ACLY species. This result indicates that ACLY acetylation and ubiquitylation are mutually exclusive and is consistent with the model that K540, K546, and K554 are the sites of both ubiquitylation and acetylation. Therefore, acetylation of these lysines would block ubiquitylation.

We also found that glucose upregulates ACLY acetylation at 3K and decreases its ubiquitylation (Figure S2B). High glucose (25 mM) effectively decreased ACLY ubiquitylation, while inhibition of deacetylases clearly diminished its ubiquitylation (Figure 2E). We conclude that acetylation and ubiquitylation occur mutually exclusively at K540, K546, and K554 and that high-glucose-induced acetylation at these three sites blocks ACLY ubiquitylation and degradation.

UBR4 Targets ACLY for Degradation

UBR4 was identified as a putative ACLY-interacting protein by affinity purification coupled with mass spectrometry analysis (data not shown). To address if UBR4 is a potential ACLY E3 ligase, we determined the interaction between ACLY and UBR4 and found that ACLY interacted with the E3 ligase domain of UBR4; this interaction was enhanced by MG132 treatment (Figure 3A). UBR4 knockdown in A549 cells resulted in an increase of endogenous ACLY protein level (Figure 3C). Moreover, UBR4 knockdown significantly stabilized ACLY (Figure 3D) and decreased ACLY ubiquitylation (Figure 3E). Taken together, these results indicate that UBR4 is an ACLY E3 ligase that responds to glucose regulation.

Figure 3  UBR4 Is the E3 Ligase of ACLY

PCAF Acetylates ACLY

PCAF knockdown significantly reduced acetylation of 3K, indicating that PCAF is a potential 3K acetyltransferase in vivo (Figure 4C, upper panel). Furthermore, PCAF knockdown decreased the steady-state level of endogenous ACLY, but not ACLY mRNA (Figure 4C, middle and lower panels). Moreover, we found that PCAF knockdown destabilized ACLY (Figure 4D). In addition, overexpression of PCAF decreases ACLY ubiquitylation (Figure 4E), while PCAF inhibition increases the interaction between UBR4 E3 ligase domain and wild-type ACLY, but not 3KR (Figure 4F). Together, our results indicate that PCAF increases ACLY protein level, possibly via acetylating ACLY at 3K.

Figure 4  PCAF Is the Acetylase of ACLY

SIRT2 Deacetylates ACLY

Figure 5  SIRT2 Decreases ACLY Acetylation and Increases Its Protein Levels In Vivo

Acetylation of ACLY Promotes Cell Proliferation and De Novo Lipid Synthesis

The protein levels of ACLY 3KQ and 3KR were accumulated to a level higher than the wild-type cells upon extended culture in low-glucose medium (Figure S6A, right panel), indicating a growth advantage conferred by ACLY stabilization resulting from the disruption of both acetylation and ubiquitylation at K540, K546, and K554. Cellular acetyl-CoA assay showed that cells expressing 3KQ or 3KR mutant ACLY produce more acetyl-CoA than cells expressing the wild-type ACLY under low glucose (Figures 6B and S6B), further supporting the conclusion that 3KQ or 3KR mutation stabilizes ACLY.

Figure 6  Acetylation of ACLY at 3K Promotes Lipogenesis and Tumor Cell Proliferation

ACLY is a key enzyme in de novo lipid synthesis. Silencing ACLY inhibited the proliferation of multiple cancer cell lines, and this inhibition can be partially rescued by adding extra fatty acids or cholesterol into the culture media (Zaidi et al., 2012). This prompted us to measure extracellular lipid incorporation in A549 cells after knockdown and ectopic expression of ACLY. We found that when cultured in low glucose (2.5 mM), cells expressing wild-type ACLY uptake significantly more phospholipids compared to cells expressing 3KQ or 3KR mutant ACLY (Figures 6C, 6D, and S6D). When cultured in the presence of high glucose (25 mM), however, cells expressing either the wild-type, 3KQ, or 3KR mutant ACLY all have reduced, but similar, uptake of extracellular phospholipids (Figures 6C, 6D, and S6D). The above results are consistent with a model that acetylation of ACLY induced by high glucose increases its stability and stimulates de novo lipid synthesis.

3K Acetylation of ACLY Is Increased in Lung Cancer

ACLY is reported to be upregulated in human lung cancer (Migita et al., 2008). Many small chemicals targeting ACLY have been designed for cancer treatment (Zu et al., 2012). The finding that 3KQ or 3KR mutant increased the ability of ACLY to support A549 lung cancer cell proliferation prompted us to examine 3K acetylation in human lung cancers. We collected a total of 54 pairs of primary human lung cancer samples with adjacent normal lung tissues and performed immunoblotting for ACLY protein levels. This analysis revealed that, when compared to the matched normal lung tissues, 29 pairs showed a significant increase of total ACLY protein using b-actin as a loading control (Figures 7A and S7A). The tumor sample analyses demonstrate that ACLY protein levels are elevated in lung cancers, and 3K acetylation positively correlates with the elevated ACLY protein. These data also indicate that ACLY with 3K acetylation may be potential biomarker for lung cancer diagnosis.

Figure 7
  Acetylation of ACLY at 3K Is Upregulated in Human Lung Carcinoma

Dysregulation of cellular metabolism is a hallmark of cancer (Hanahan and Weinberg, 2011Vander Heiden et al., 2009). Besides elevated glycolysis, increased lipogenesis, especially de novo lipid synthesis, also plays an important role in tumor growth. Because most carbon sources for fatty acid synthesis are from glucose in mammalian cells (Wellen et al., 2009), the channeling of carbon into de novo lipid synthesis as building blocks for tumor cell growth is primarily linked to acetyl-CoA production by ACLY. Moreover, the ACLY-catalyzed reaction consumes ATP. Therefore, as the key cellular energy and carbon source, one may expect a role for glucose in ACLY regulation. In the present study, we have uncovered a mechanism of ACLY regulation by glucose that increases ACLY protein level to meet the enhanced demand of lipogenesis in growing cells, such as tumor cells (Figure 7C). Glucose increases ACLY protein levels by stimulating its acetylation.

Upregulation of ACLY is common in many cancers (Kuhajda, 2000Milgraum et al., 1997Swinnen et al., 2004Yahagi et al., 2005). This is in part due to the transcriptional activation by SREBP-1 resulting from the activation of the PI3K/AKT pathway in cancers (Kim et al., 2010Nadler et al., 2001Wang and Dey, 2006). In this study, we report a mechanism of ACLY regulation at the posttranscriptional level. We propose that acetylation modulated by glucose status plays a crucial role in coordinating the intracellular level of ACLY, hence fatty acid synthesis, and glucose availability. When glucose is sufficient, lipogenesis is enhanced. This can be achieved, at least in part, by the glucose-induced stabilization of ACLY. High glucose increases ACLY acetylation, which inhibits its ubiquitylation and degradation, leading to the accumulation of ACLY and enhanced lipogenesis. In contrast, when glucose is limited, ACLY is not acetylated and thus can be ubiquitylated, leading to ACLY degradation and reduced lipogenesis. Moreover, our data indicate that acetylation and ubiquitylation in ACLY may compete with each other by targeting the same lysine residues at K540, K546, and K554. Consistently, previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). Similar models of different modifications on the same lysine residues have been reported in the regulation of other proteins (Grönroos et al., 2002Li et al., 20022012). We propose that acetylation and ubiquitylation have opposing effects in the regulation of ACLY by competitively modifying the same lysine residues. The acetylation-mimetic 3KQ and the acetylation-deficient 3KR mutants behaved indistinguishably in most biochemical and functional assays, mainly due to the fact that these mutations disrupt lysine ubiquitylation that primarily occurs on these three residues.

ACLY is increased in lung cancer tissues compared to adjacent tissues. Consistently, ACLY acetylation at 3K is also significantly increased in lung cancer tissues. These observations not only confirm ACLY acetylation in vivo, but also suggest that ACLY 3K acetylation may play a role in lung cancer development. Our study reveals a mechanism of ACLY regulation in response to glucose signals.

 

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Nomura DK1Long JZNiessen SHoover HSNg SWCravatt BF.
Cell. 2010 Jan 8; 140(1):49-61
http://dx.doi.org/10.1016.2Fj.cell.2009.11.027

Highlights

  • Monoacylglycerol lipase (MAGL) is elevated in aggressive human cancer cells
  • Loss of MAGL lowers fatty acid levels in cancer cells and impairs pathogenicity
  • MAGL controls a signaling network enriched in protumorigenic lipids
  • A high-fat diet can restore the growth of tumors lacking MAGL in vivo
monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

http://www.cell.com/cms/attachment/1082768/7977146/fx1.jpg

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity—phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.

We show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in non-aggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity — phenotypes that are reversed by an MAGL inhibitor. Interestingly, impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of pro-tumorigenic signals.

The conversion of cells from a normal to cancerous state is accompanied by reprogramming of metabolic pathways (Deberardinis et al., 2008Jones and Thompson, 2009Kroemer and Pouyssegur, 2008), including those that regulate glycolysis (Christofk et al., 2008Gatenby and Gillies, 2004), glutamine-dependent anaplerosis (DeBerardinis et al., 2008DeBerardinis et al., 2007Wise et al., 2008), and the production of lipids (DeBerardinis et al., 2008Menendez and Lupu, 2007). Despite a growing appreciation that dysregulated metabolism is a defining feature of cancer, it remains unclear, in many instances, how such biochemical changes occur and whether they play crucial roles in disease progression and malignancy.

Among dysregulated metabolic pathways, heightened de novo lipid biosynthesis, or the development a “lipogenic” phenotype (Menendez and Lupu, 2007), has been posited to play a major role in cancer. For instance, elevated levels of fatty acid synthase (FAS), the enzyme responsible for fatty acid biosynthesis from acetate and malonyl CoA, are correlated with poor prognosis in breast cancer patients, and inhibition of FAS results in decreased cell proliferation, loss of cell viability, and decreased tumor growth in vivo (Kuhajda et al., 2000Menendez and Lupu, 2007Zhou et al., 2007). FAS may support cancer growth, at least in part, by providing metabolic substrates for energy production (via fatty acid oxidation) (Buzzai et al., 2005Buzzai et al., 2007Liu, 2006). Many other features of lipid biochemistry, however, are also critical for supporting the malignancy of cancer cells, including:

Prominent examples of lipid messengers that contribute to cancer include:

Here, we use functional proteomic methods to discover a lipolytic enzyme, monoacylglycerol lipase (MAGL), that is highly elevated in aggressive cancer cells from multiple tissues of origin. We show that MAGL, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells. The resulting MAGL-FFA pathway feeds into a diverse lipid network enriched in pro-tumorigenic signaling molecules and promotes migration, survival, and in vivo tumor growth. Aggressive cancer cells thus pair lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behavior.

Activity-Based Proteomic Analysis of Hydrolytic Enzymes in Human Cancer Cells

To identify enzyme activities that contribute to cancer pathogenesis, we conducted a functional proteomic analysis of a panel of aggressive and non-aggressive human cancer cell lines from multiple tumors of origin, including melanoma [aggressive (C8161, MUM2B), non-aggressive (MUM2C)], ovarian [aggressive (SKOV3), non-aggressive (OVCAR3)], and breast [aggressive (231MFP), non-aggressive (MCF7)] cancer. Aggressive cancer lines were confirmed to display much greater in vitro migration and in vivo tumor-growth rates compared to their non-aggressive counterparts (Figure S1), as previously shown (Jessani et al., 2004;Jessani et al., 2002Seftor et al., 2002Welch et al., 1991). Proteomes from these cancer lines were screened by activity-based protein profiling (ABPP) using serine hydrolase-directed fluorophosphonate (FP) activity-based probes (Jessani et al., 2002Patricelli et al., 2001). Serine hydrolases are one of the largest and most diverse enzyme classes in the human proteome (representing ~ 1–1.5% of all human proteins) and play important roles in many biochemical processes of potential relevance to cancer, such as proteolysis (McMahon and Kwaan, 2008Puustinen et al., 2009), signal transduction (Puustinen et al., 2009), and lipid metabolism (Menendez and Lupu, 2007Zechner et al., 2005). The goal of this study was to identify hydrolytic enzyme activities that were consistently altered in aggressive versus non-aggressive cancer lines, working under the hypothesis that these conserved enzymatic changes would have a high probability of contributing to the pathogenic state of cancer cells.

Among the more than 50 serine hydrolases detected in this analysis (Tables S13), two enzymes, KIAA1363 and MAGL, were found to be consistently elevated in aggressive cancer cells relative to their non-aggressive counterparts, as judged by spectral counting (Jessani et al., 2005Liu et al., 2004). We confirmed elevations in KIAA1363 and MAGL in aggressive cancer cells by gel-based ABPP, where proteomes are treated with a rhodamine-tagged FP probe and resolved by 1D-SDS-PAGE and in-gel fluorescence scanning (Figure 1A). In both cases, two forms of each enzyme were detected (Figure 1A), due to differential glycoslyation for KIAA1363 (Jessani et al., 2002), and possibly alternative splicing for MAGL (Karlsson et al., 2001). We have previously shown that KIAA1363 plays a role in regulating ether lipid signaling pathways in aggressive cancer cells (Chiang et al., 2006). On the other hand, very little was known about the function of MAGL in cancer.

Figure 1  MAGL is elevated in aggressive cancer cells, where the enzyme regulates monoacylgycerol (MAG) and free fatty acid (FFA) levels

The heightened activity of MAGL in aggressive cancer cells was confirmed using the substrate C20:4 MAG (Figure 1B). Since several enzymes have been shown to display MAG hydrolytic activity (Blankman et al., 2007), we confirmed the contribution that MAGL makes to this process in cancer cells using the potent and selective MAGL inhibitor JZL184 (Long et al., 2009a).

MAGL Regulates Free Fatty Acid Levels in Aggressive Cancer Cells

MAGL is perhaps best recognized for its role in degrading the endogenous cannabinoid 2-arachidonoylglycerol (2-AG, C20:4 MAG), as well as other MAGs, in brain and peripheral tissues (Dinh et al., 2002Long et al., 2009aLong et al., 2009bNomura et al., 2008). Consistent with this established function, blockade of MAGL by JZL184 (1 μM, 4 hr) produced significant elevations in the levels of several MAGs, including 2-AG, in each of the aggressive cancer cell lines (Figure 1C and Figure S2). Interestingly, however, MAGL inhibition also caused significant reductions in the levels of FFAs in aggressive cancer cells (Figure 1D and Figure S2). This surprising finding contrasts with the function of MAGL in normal tissues, where the enzyme does not, in general, control the levels of FFAs (Long et al., 2009aLong et al., 2009b;Nomura et al., 2008).

Metabolic labeling studies using the non-natural C17:0-MAG confirmed that MAGs are converted to LPC and LPE by aggressive cancer cells, and that this metabolic transformation is significantly enhanced by treatment with JZL184 (Figure S1). Finally, JZL184 treatment did not affect the levels of MAGs and FFAs in non-aggressive cancer lines (Figure 1C, D), consistent with the negligible expression of MAGL in these cells (Figure 1A, B).

We next stably knocked down MAGL expression by RNA interference technology using two independent shRNA probes (shMAGL1, shMAGL2), both of which reduced MAGL activity by 70–80% in aggressive cancer lines (Figure 2A, D and Figure S2). Other serine hydrolase activities were unaffected by shMAGL probes (Figure 2A, D and Figures S2), confirming the specificity of these reagents. Both shMAGL probes caused significant elevations in MAGs and corresponding reductions in FFAs in aggressive melanoma (Figure 2B, C), ovarian (Figure 2E, F), and breast cancer cells (Figure S2).

Figure 2  Stable shRNA-mediated knockdown of MAGL lowers FFA levels in aggressive cancer cells.

Together, these data demonstrate that both acute (pharmacological) and stable (shRNA) blockade of MAGL cause elevations in MAGs and reductions in FFAs in aggressive cancer cells. These intriguing findings indicate that MAGL is the principal regulator of FFA levels in aggressive cancer cells. Finally, we confirmed that MAGL activity (Figure 3A, B) and FFA levels (Figure 3C) are also elevated in high-grade primary human ovarian tumors compared to benign or low-grade tumors. Thus, heightened expression of the MAGL-FFA pathway is a prominent feature of both aggressive human cancer cell lines and primary tumors.

Figure 3  High-grade primary human ovarian tumors possess elevated MAGL activity and FFAs compared to benign tumors.

Disruption of MAGL Expression and Activity Impairs Cancer Pathogenicity

shMAGL cancer lines were next examined for alterations in pathogenicity using a set of in vitro and in vivo assays. shMAGL-melanoma (C8161), ovarian (SKOV3), and breast (231MFP) cancer cells exhibited significantly reduced in vitro migration (Figure 4A, F and Figure S2), invasion (Figure 4B, G and Figure S2), and cell survival under serum-starvation conditions (Figure 4C, H and Figure S2). Acute pharmacological blockade of MAGL by JZL184 also decreased cancer cell migration (Figure S2), but not survival, possibly indicating that maximal impairments in cancer aggressiveness require sustained inhibition of MAGL.

Figure 4  shRNA-mediated knockdown and pharmacological inhibition of MAGL impair cancer aggressiveness.

MAGL Overexpression Increases FFAs and the Aggressiveness of Cancer Cells

Stable MAGL-overexpressing (MAGL-OE) and control [expressing an empty vector or a catalytically inactive version of MAGL, where the serine nucleophile was mutated to alanine (S122A)] variants of MUM2C and OVCAR3 cells were generated by retroviral infection and evaluated for their respective MAGL activities by ABPP and C20:4 MAG substrate assays. Both assays confirmed that MAGL-OE cells possess greater than 10-fold elevations in MAGL activity compared to control cells (Figure 5A and Figure S4). MAGL-OE cells also showed significant reductions in MAGs (Figure 5B andFigure S4) and elevated FFAs (Figure 5C and Figure S4). This altered metabolic profile was accompanied by increased migration (Figure 5D and Figure S4), invasion (Figure 5E and Figure S4), and survival (Figure S4) in MAGL-OE cells. None of these effects were observed in cancer cells expressing the S122A MAGL mutant, indicating that they require MAGL activity.  MAGL-OE MUM2C cells also showed enhanced tumor growth in vivo compared to control cells (Figure 5F). Notably, the increased tumor growth rate of MAGL-OE MUM2C cells nearly matched that of aggressive C8161 cells (Figure S4). These data indicate that the ectopic expression of MAGL in non-aggressive cancer cells is sufficient to elevate their FFA levels and promote pathogenicity both in vitro and in vivo.

Figure 5 Ectopic expression of MAGL elevates FFA levels and enhances the in vitro and in vivo pathogenicity of MUM2C melanoma cells.

Metabolic Rescue of Impaired Pathogenicity in MAGL-Disrupted Cancer Cells

MAGL could support the aggressiveness of cancer cells by either reducing the levels of its MAG substrates, elevating the levels of its FFA products, or both. Among MAGs, the principal signaling molecule is the endocannabinoid 2-AG, which activates the CB1 and CB2 receptors (Ahn et al., 2008Mackie and Stella, 2006). The endocannabinoid system has been implicated previously in cancer progression and, depending on the specific study, shown to promote (Sarnataro et al., 2006Zhao et al., 2005) or suppress (Endsley et al., 2007Wang et al., 2008) cancer pathogenesis. Neither a CB1 or CB2 antagonist rescued the migratory defects of shMAGL cancer cells (Figure S5). CB1 and CB2 antagonists also did not affect the levels of MAGs or FFAs in cancer cells (Figure S5).

We then determined whether increased FFA delivery could rectify the tumor growth defect observed for shMAGL cells in vivo. Immune-deficient mice were fed either a normal chow or high-fat diet throughout the duration of a xenograft tumor growth experiment. Notably, the impaired tumor growth rate of shMAGL-C8161 cells was completely rescued in mice fed a high-fat diet. In contrast, shControl-C8161 cells showed equivalent tumor growth rates on a normal versus high-fat diet. The recovery in tumor growth for shMAGL-C8161 cells in the high-fat diet group correlated with significantly increases levels of FFAs in excised tumors (Figure 6D). Collectively, these results indicate that MAGL supports the pathogenic properties of cancer cells by maintaining tonically elevated levels of FFAs.

Figure 6  Recovery of the pathogenic properties of shMAGL cancer cells by treatment with exogenous fatty acids.

MAGL Regulates a Fatty Acid Network Enriched in Pro-Tumorigenic Signals

Studies revealed that neither

  • the MAGL-FFA pathway might serve as a means to regenerate NAD+ (via continual fatty acyl glyceride/FFA recycling) to fuel glycolysis, or
  • increased lipolysis could be to generate FFA substrates for β-oxidation, which may serve as an important energy source for cancer cells (Buzzai et al., 2005), or
  • CPT1 blockade (reduced expression of CPT1 in aggressive cancer cells (data not shown) has been reported previously (Deberardinis et al., 2006))

providing evidence against a role for β-oxidation as a downstream mediator of the pathogenic effects of the MAGL-fatty acid pathway.

Considering that FFAs are fundamental building blocks for the production and remodeling of membrane structures and signaling molecules, perturbations in MAGL might be expected to affect several lipid-dependent biochemical networks important for malignancy. To test this hypothesis, we performed lipidomic analyses of cancer cell models with altered MAGL activity, including comparisons of:

  1. MAGL-OE versus control cancer cells (OVCAR3, MUM2C), and
  2. shMAGL versus shControl cancer cells (SKOV3, C8161).

Complementing these global profiles, we also conducted targeted measurements of specific bioactive lipids (e.g., prostaglandins) that are too low in abundance for detection by standard lipidomic methods. The resulting data sets were then mined to identify a common signature of lipid metabolites regulated by MAGL, which we defined as metabolites that were significantly increased or reduced in MAGL–OE cells and showed the opposite change in shMAGL cells relative to their respective control groups (Figure 7A, B and Table S4).

Figure 7  MAGL regulates a lipid network enriched in pro-tumorigenic signaling molecules.

Most of the lipids in the MAGL-fatty acid network, including several lysophospholipids (LPC, LPA, LPE), ether lipids (MAGE, alkyl LPE), phosphatidic acid (PA), and prostaglandin E2 (PGE2), displayed similar profiles to FFAs, being consistently elevated and reduced in MAGL-OE and shMAGL cells, respectively. Only MAGs were found to show the opposite profile (elevated and reduced in shMAGL and MAGL-OE cells, respectively). Interestingly, virtually this entire lipidomic signature was also observed in aggressive cancer cells when compared to their non-aggressive counterparts (e.g., C8161 versus MUM2C and SKOV3 versus OVCAR3, respectively; Table S4). These findings demonstrate that MAGL regulates a lipid network in aggressive cancer cells that consists of not only FFAs and MAGs, but also a host of secondary lipid metabolites. Increases (rather than decreases) in LPCs and LPEs were observed in JZL184-treated cells (Figure S1 and Table S4). These data indicate that acute and chronic blockade of MAGL generate distinct metabolomic effects in cancer cells, likely reflecting the differential outcomes of short- versus long-term depletion of FFAs.

Within the MAGL-fatty acid network are several pro-tumorigenic lipid messengers, including LPA and PGE2, that have been reported to promote the aggressiveness of cancer cells (Gupta et al., 2007Mills and Moolenaar, 2003). Metabolic labeling studies confirmed that aggressive cancer cells can convert both MAGs and FFAs (Figure S1) to LPA and PGE2 and, for MAGs, this conversion was blocked by JZL184 (Figure S1). Interestingly, treatment with either LPA or PGE2 (100 nM, 4 hr) rescued the impaired migration of shMAGL cancer cells at concentrations that did not affect the migration of shControl cells (Figure 7E).

Heightened lipogenesis is an established early hallmark of dysregulated metabolism and pathogenicity in cancer (Menendez and Lupu, 2007). Cancer lipogenesis appears to be driven principally by FAS, which is elevated in most transformed cells and important for survival and proliferation (De Schrijver et al., 2003;Kuhajda et al., 2000Vazquez-Martin et al., 2008). It is not yet clear how FAS supports cancer growth, but most of the proposed mechanisms invoke pro-tumorigenic functions for the enzyme s fatty acid products and their lipid derivatives (Menendez and Lupu, 2007). This creates a conundrum, since the fatty acid molecules produced by FAS are thought to be rapidly incorporated into neutral- and phospho-lipids, pointing to the need for complementary lipolytic pathways in cancer cells to release stored fatty acids for metabolic and signaling purposes (Prentki and Madiraju, 2008Przybytkowski et al., 2007). Consistent with this hypothesis, we found that acute treatment with the FAS inhibitor C75 (40 μM, 4 h) did not reduce FFA levels in cancer cells (data not shown). Furthermore, aggressive and non-aggressive cancer cells exhibited similar levels of FAS (data not shown), indicating that lipogenesis in the absence of paired lipolysis may be insufficient to confer high levels of malignancy.

Here we show that aggressive cancer cells do indeed acquire the ability to liberate FFAs from neutral lipid stores as a consequence of heightened expression of MAGL. MAGL and its FFA products were found to be elevated in aggressive human cancer cell lines from multiple tissues of origin, as well as in high-grade primary human ovarian tumors. These data suggest that the MAGL-FFA pathway may be a conserved feature of advanced forms of many types of cancer. Further evidence in support of this premise originates from gene expression profiling studies, which have identified increased levels of MAGL in primary human ductal breast tumors compared to less malignant medullary breast tumors (Gjerstorff et al., 2006). The key role that MAGL plays in regulating FFA levels in aggressive cancer cells contrasts with the function of this enzyme in normal tissues, where it mainly controls the levels of MAGs, but not FFAs (Long et al., 2009b). These data thus provide a striking example of the co-opting of an enzyme by cancer cells to serve a distinct metabolic purpose that supports their pathogenic behavior.

Taken together, our results indicate that MAGL serves as key metabolic hub in aggressive cancer cells, where the enzyme regulates a fatty acid network that feeds into a number of pro-tumorigenic signaling pathways.

 

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.8.1  Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling

Komai K1Niwa Y1Sasazawa Y1Simizu S2.
FEBS Lett. 2015 Mar 12; 589(6):738-43
http://dx.doi.org:/10.1016/j.febslet.2015.01.040

Highlights

  • Pirin decreases E-cadherin expression and induces EMT.
  • The induction of EMT by Pirin is achieved through a Bcl3 independent pathway.
  • Pirin may be a novel target for cancer therapy.

Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E-cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle-like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E-cadherin expression similar to wild-type, suggesting that Pirin regulates E-cadherin independently of Bcl3-Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.

Pirin regulates the expression of E-cadherin and EMT markers

In melanoma, Pirin enhances NF-jB activity and increases Slug expression by binding Bcl3 [31], and it may also be involved in adenoid cystic tumor metastasis [23]. Since Slug suppresses E-cadherin transcription and is recognized as a major EMT inducer, we hypothesized that Pirin may regulate EMT through inducing Slug expression. To investigate whether Pirin regulates EMT, we measured E-cadherin expression following Pirin knockdown. As shown in Fig. 1A and B, E-cadherin expression was significantly increased following Pirin knockdown indicating that it may promote EMT. To confirm this, we established Pirin-expressing HeLa cells (Fig. 1C), which inhibited the expression of E-cadherin (Fig. 1D). Additionally, the expression of Occludin, an epithelial marker, was decreased, and several mesenchymal markers, including Fibronectin, N-cadherin, and Vimentin, were increased by Pirin expression (Fig. 1D). These data suggest that Pirin promotes EMT.

Pirin induces EMT-associated cell morphological changes

As mentioned above, cells undergo morphological changes during EMT. Therefore, we next analyzed whether Pirin expression affects cell morphology. Quantitative analysis of morphological changes was based on cell circularity, {4p(area)/(perimeter)2}100, which decreases during EMT-associated morphological changes [34–36]. Indeed, TGF-b or TNF-a exposure induced EMTassociated cell morphological changes in HeLa cells (data not shown). Employing this parameter of circularity, we compared the morphology of our established HeLa/Pirin-GFP cells with control HeLa/GFP cells. Although the control HeLa/GFP cells displayed a cobblestone-like morphology, HeLa/Pirin-GFP cells were elongated in shape (Fig. 2A). Indeed, compared with control cells, the circularity of HeLa/Pirin-GFP cells was significantly decreased (Fig. 2B). To confirm that these observations were dependent on Pirin expression, HeLa/Pirin-GFP cells were treated with an siRNA targeting Pirin. HeLa/Pirin-GFP cells recovered a cobblestone-like morphology (Fig. 2C) and circularity (Fig. 2D) when treated with Pirin siRNA indicating that Pirin expression induces EMT.

Pirin induces cell migration

During EMT cells acquire migratory capabilities. Therefore, we analyzed whether Pirin affects cell migration. HeLa cells were treated with an siRNA targeting Pirin and migration was assessed using a wound healing assay. Although Pirin knockdown had no effect on cell proliferation (data not shown), wound repair was inhibited in Pirin-depleted HeLa cells (Fig. 3A and B) suggesting that Pirin promoted cell migration. Furthermore, camptothecin treatment of HeLa/GFP cells caused decreased cell viability in a dose-dependent manner, whereas HeLa/Pirin-GFP cells were more resistantto drugtreatment (datanot shown).These results suggest that Pirin induces EMT-like phenotypes, such as cell migration and anticancer drug resistance.
Pirin regulates EMT independently of Bcl3-Slug signaling

To investigate whether Pirin controls E-cadherin expression at the transcriptional level, we measured E-cadherin promoter activity with a reporter assay. Indeed, the luciferase reporter analysis indicated that Pirin inhibited E-cadherin promoter activity (Fig. 4A and B). To determine if Bcl3 is involved in Pirin-induced EMT, we tested whether a Pirin mutant defective in Bcl3 binding could inhibit E-cadherin expression. We generated a mutation in the metal-binding cavity of Pirin(E103A) and confirmed that it disrupted Bcl3 binding. In vitro GST pull-down analysis using recombinant Pirin and Bcl3/ARD demonstrated that the Pirin mutant was defective for Bcl3 binding compared to wild-type (Fig. 5A). Interestingly, expression of both wild-type Pirin and the mutant defective in Bcl3 binding reduced E-cadherin gene and protein expression (Fig. 5B and C). Taken together these results indicate that Pirin decreases E-cadherin expression without binding Bcl3, and suggest that Pirin regulates EMT independently of Bcl3-Slug signaling.

Discussion

A characteristic feature of EMT is the disruption of epithelial cell–cell contact, which is achieved by reduced E-cadherin expression. Therefore, revealing the regulatory pathways controlling E-cadherin expression may elucidate the mechanisms of EMT. Several transcription factors regulate E-cadherin transcription. For instance,Snail,Slug,Twist,and Zebact as mastertranscriptional regulators that bind the consensus E-box sequence in the E-cadherin gene promoter and decrease the transcriptional activity [38]. Since Pirin regulates the transcription of Slug [31], we hypothesized that Pirin may also regulate EMT. In this study we demonstrated that Pirin decreases E-cadherin expression, and induces EMT and cancer malignant phenotypes. Since EMT is an initial step of metastasis, Pirin may contribute to cancer progression. We next examined whether the regulation of EMT by Pirin is attributed to Bcl3 binding and the induction of Slug. To this end, we generated a Pirin mutant (E103A) defective for Bcl3 binding (Fig. 5A). Single Fe2+ ion chelating is coordinated by His56, His58, His101, and Glu103 of Pirin, and the N-terminal domain containing these residues is highly conserved between mammals, plants, fungi, and prokaryotic organisms [15,27]. Therefore, it has been predicted that this N-terminal domain containing the metal-binding cavity is important for Pirin function [20,26,31]. Indeed, TPh A inserts into the metal-binding cavity and inhibits binding to Bcl3 suggesting that the interaction occurs with the metal-binding cavity of Pirin [31]. In contrast, Hai Pang suggests that a Pirin–Bcl3– (p50)2 complex forms between acidic regions of the N-terminal Pirin domain at residues 77–82, 97–103 and 124–128 with a basic patch of Bcl3 [27]. In this study, we mutated Glutamic acid 103, a residue common between Hai Pang’s model and Pirin’s metalbinding cavity. Pull-down analysis indicated that an E103A mutant is defectiveinfor Bcl3binding(Fig.5A). Thisis the firstexperimental demonstration showing that Glu103 of Pirin is important Bcl3 binding. However, expression of the E103A mutant suppressed Ecadherin gene expression similarly to wild-type Pirin (Fig. 5B and C). Although the Bcl3–(p50)2 complex participates in oncogene addiction in cervical cells [39,40], expression of Pirin in HeLa cells did not increase Slug expression (data not shown). Therefore, we concludethatPirindecreasesE-cadherinexpressionindependently of Bcl3-Slug signaling. To understand how Pirin suppresses E-cadherin gene expression, we analyzed E-cadherin promoter activity (Fig. 4). Since Pirin decreased the activity of the E-cadherin promoter (995+1), we constructed a series of promoter deletion mutants (795+1, 565+1, 365+1, 175+1) to identify a region important for Pirin-mediated regulation. Expression of Pirin decreased the transcriptional activity of all constructs (Supplementary Fig. S1A), suggesting that Pirin may suppress E-cadherin expression through element(s) in region 175+1. Yan-Nan Liu and colleagues proposed that this region contains four Sp1-binding sites and two E-boxes that regulate E-cadherin expression.

Fig. 1. Pirin regulates E-cadherin gene expression. (A, B) HeLa cells were transfected with siRNA targeting Pirin (siPirin#1 or #2) or control siRNA (siCTRL). Forty-eight hours after transfection, cDNA was used for PCR using primer sets specific against Pirin, E-cadherin and GAPDH (A). Forty-eight hours after transfection, HeLa cells were lysed and the lysates were analyzed by Western blot with the indicated antibodies (B). (C) Lysates from HeLa/Pirin-GFP and HeLa/GFP cells were analyzed by Western blot with the indicated antibodies. (D) cDNA from HeLa/GFP or HeLa/Pirin-GFP cells was used for PCR to determine the effect of Pirin on the expression of EMT marker genes.

Fig. 2. Pirin induces cell morphological changes associated with EMT. (A) Phase contrast and fluorescence microscopic images were taken of HeLa/GFP and HeLa/Pirin-GFP cells. (B) Cell circularity was defined as form factor, {4p(area)/(perimeter)2}100 [%], and calculated using Image J software. A random selection of 100 cells from each condition was measured. (C, D) Phase contrast and fluorescence microscopic images were taken of siRNA-treated HeLa/GFP and HeLa/Pirin-GFP cells. Each cell line was transfected with siPirin#2 or siCTRL. Cells were observed by microscopy 48 h after transfection (C) and circularity was measured (D). Data shown are means ± s.d. ⁄P <0.05, bars 100lm.

Fig. 3. Pirin knockdown suppresses cell migration. (A, B) HeLa cells were transfected with siPirin#2 or siCTRL. An artificial wound was created with a tip 24h after transfection and cells were cultured for an additional 12 h. For quantification, the cells were photographed after 12h of incubation (A) and the area covered by cells was measured using Image J and normalized to control cells (B).

Fig. 4. Pirin regulates E-cadherin promoter activity.(A). HeLacells were transfected with siPirin#2 or siGFP (control) and cultured for 24 h. The E-cadherin promoter construct (995+1) and phRL-TK vectorwere transfected and cellswere cultured for an additional 24 h. Luciferase activities were measured and normalized to Renilla luciferase activity. (B) HeLa cells were transfected with the promoter construct (995+1), phRL-TK vector, and a Pirin expression vector. After 24 h, luciferase activities were measured and normalized to Renilla luciferase activity. Data are the mean ± s.d. ⁄P < 0.05.

Fig. 5. Pirin decreases E-cadherin expression in a Bcl3-independent manner. (A) Purified His6-Pirin and His6-Pirin(E103A) were incubated with Glutathione-Sepharose beads conjugated to GST or GST-Bcl3/ARD. The samples were analyzed by Western blot. (B, C) HeLa cells were transfected with vectors encoding GFP, Pirin-GFP, or Pirin(E103A)GFP. Cells were lysed 48 h after transfection and lysates were analyzed by Western blot (B). RNA collected at 48h was used for RT-PCR with the specified primer sets for each gene (C).

7.7.8.2 1324 PIRIN DOWN-REGULATES THE EAF2/U19 SIGNALING AND RETARDS THE GROWTH INHIBITION INDUCED BY EAF2/U19 IN PROSTATE CANCER CELLS

Zhongjie Qiao, Dan Wang, Zhou Wang
The Journal of Urology Apr 2013; 189(4), Supplement: e541
http://dx.doi.org/10.1016/j.juro.2013.02.2678
EAF2/U19, as the tumor suppressor, has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth in vivo, and its expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification and characterization of the binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells.

7.7.8.3 Pirin Inhibits Cellular Senescence in Melanocytic Cells

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression

Cellular senescence is a physiological process through which normal somatic cells lose their ability to divide and enter an irreversible state of cell cycle arrest, although they remain viable and metabolically active.1,2The specific molecular circuitry underlying the onset of cellular senescence is dependent on the type of stimulus and on the cellular context. A central role is held by the activation of the tumor suppressor proteins p53 and retinoblastoma susceptibility protein (pRB),3–5 which act by interfering with the transcriptional program of the cell and ultimately arresting cell cycle progression.

In the last decade, senescence has been recognized as a major barrier against the development of tumors in mammals.6–8 One of the most prominent in vivo examples is represented by nevi, in which cells proliferate after oncogene activation and then become senescent. Melanoma is a highly aggressive form of neoplasm often observed to derive from nevi, and the transition implies suppression of the mechanisms that sustain the onset and maintenance of senescence.9 In fact, many of the melanoma-associated tumor suppressor genes identified to date are themselves involved in control of senescence, including BRAF (encoding serine/threonine-protein kinase B-raf), CKD4 (cyclin-dependent kinase 4), and CDKN2A (encoding cyclin-dependent kinase inhibitor 2A isoforms p16INK4a and p19ARF).3,10

Nevi frequently harbor oncogenic mutations of the tyrosine kinase BRAF gene, particularly V600E,11 andBRAFV600E is also found in approximately 70% of cutaneous melanomas.12 Expression of BRAFV600E in human melanocytes leads to oncogene-induced senescence,8 which can be considered as a mechanism that protects from malignant progression. In time, some cells may eventually escape senescence, probably through the acquisition of additional genetic abnormalities, thus favoring transformation to melanoma.13

Pirin (PIR) is a highly conserved nuclear protein belonging to the Cupin superfamily14 whose function is, to date, poorly characterized. It has been described as a putative transcriptional regulator on the basis of its physical association with the nuclear I/CCAAT box transcription factor NFI/CTF115 and with the B-cell lymphoma protein, BCL-3, a regulator of NF-κB/Rel activity. A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG).16 Other reports described quercetinase enzymatic activity,17 and regulation of apoptosis18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.

There is evidence of variations in PIR expression levels in different types of malignancies, but a systematic analysis of PIR expression in human tumors has been lacking. We analyzed PIR expression pattern in a collection of normal and neoplastic human tissues and found that it is expressed in scattered melanocytes, virtually absent in more mature regions of nevi, and present at high levels in a subset of melanomas. Functional studies performed in normal and transformed melanocytic cells revealed that PIR ablation results in cellular senescence, and that PIR levels decrease in response to senescence stimuli. Our results suggest that PIR may be a relevant player in the negative control of cellular senescence in PIR-expressing melanomas.

PIR overexpression in melanoma

Figure 3  PIR overexpression in PIR melanoma cells has no effect on proliferation.
PIR Expression Is Down-Regulated by BRAF Activation and Camptothecin Treatment

BRAF mutations are frequent in nevi, and are directly linked to the induction of oncogene-induced senescence. Variations in PIR expression levels were therefore investigated in an experimental model of senescence induced by oncogenic BRAF. Human diploid fibroblasts (TIG3–hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT).28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation (Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

Brian A. Lewis
Biochim et Biophys Acta (BBA) – Gene Regulatory Mechanisms Nov 2013; 1829(11): 1202–1206
http://dx.doi.org/10.1016/j.bbagrm.2013.09.003

Highlights

  • This review article discusses recent advances in the links between O-GlcNAc and transcriptional regulation.
  • Discusses several systems to illustrate O-GlcNAc dynamics: Tet proteins, MLL complexes, circadian clock proteins and RNA pol II.
  • Suggests that promoters are nutrient sensors.

Post-translational modifications play important roles in transcriptional regulation. Among the less understood PTMs is O-GlcNAcylation. Nevertheless, O-GlcNAcylation in the nucleus is found on hundreds of transcription factors and coactivators and is often found in a mutually exclusive ying–yang relationship with phosphorylation. O-GlcNAcylation also links cellular metabolism directly to the proteome, serving as a conduit of metabolic information to the nucleus. This review serves as a brief introduction to O-GlcNAcylation, emphasizing its important thematic roles in transcriptional regulation, and highlights several recent and important additions to the literature that illustrate the connections between O-GlcNAc and transcription.

links between O-GlcNAc and transcriptional regulation.

links between O-GlcNAc and transcriptional regulation.

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr1.sml
links between O-GlcNAc and transcriptional regulation.

systems to illustrate O-GlcNAc dynamics

systems to illustrate O-GlcNAc dynamics

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr2.sml
systems to illustrate O-GlcNAc dynamics

7.7.10 O-GlcNAcylation in cellular functions and human diseases

Yang YR1Suh PG2.
Adv Biol Regul. 2014 Jan; 54:68-73
http://dx.doi.org:/10.1016/j.jbior.2013.09.007

O-GlcNAcylation is dynamic and a ubiquitous post-translational modification. O-GlcNAcylated proteins influence fundamental functions of proteins such as protein-protein interactions, altering protein stability, and changing protein activity. Thus, aberrant regulation of O-GlcNAcylation contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, metabolic disorders, and Alzheimer’s disease. Diverse cellular signaling systems are involved in pathogenesis of these diseases. O-GlcNAcylated proteins occur in many different tissues and cellular compartments and affect specific cell signaling. This review focuses on the O-GlcNAcylation in basic cellular functions and human diseases.

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr2.sml
O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

aberrant regulation of O-GlcNAcylation in disease

aberrant regulation of O-GlcNAcylation in disease

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr3.sml
aberrant regulation of O-GlcNAcylation in disease

 Comment:

Body of review in energetic metabolic pathways in malignant T cells

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.

Dr. Aurel,
Targu Jiu

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Imaging-guided cancer treatment


Imaging-guided cancer treatment

Writer & reporter: Dror Nir, PhD

It is estimated that the medical imaging market will exceed $30 billion in 2014 (FierceMedicalImaging). To put this amount in perspective; the global pharmaceutical market size for the same year is expected to be ~$1 trillion (IMS) while the global health care spending as a percentage of Gross Domestic Product (GDP) will average 10.5% globally in 2014 (Deloitte); it will reach ~$3 trillion in the USA.

Recent technology-advances, mainly miniaturization and improvement in electronic-processing components is driving increased introduction of innovative medical-imaging devices into critical nodes of major-diseases’ management pathways. Consequently, in contrast to it’s very small contribution to global health costs, medical imaging bears outstanding potential to reduce the future growth in spending on major segments in this market mainly: Drugs development and regulation (e.g. companion diagnostics and imaging surrogate markers); Disease management (e.g. non-invasive diagnosis, guided treatment and non-invasive follow-ups); and Monitoring aging-population (e.g. Imaging-based domestic sensors).

In; The Role of Medical Imaging in Personalized Medicine I discussed in length the role medical imaging assumes in drugs development.  Integrating imaging into drug development processes, specifically at the early stages of drug discovery, as well as for monitoring drug delivery and the response of targeted processes to the therapy is a growing trend. A nice (and short) review highlighting the processes, opportunities, and challenges of medical imaging in new drug development is: Medical imaging in new drug clinical development.

The following is dedicated to the role of imaging in guiding treatment.

Precise treatment is a major pillar of modern medicine. An important aspect to enable accurate administration of treatment is complementing the accurate identification of the organ location that needs to be treated with a system and methods that ensure application of treatment only, or mainly to, that location. Imaging is off-course, a major component in such composite systems. Amongst the available solution, functional-imaging modalities are gaining traction. Specifically, molecular imaging (e.g. PET, MRS) allows the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. In oncology, it can be used to depict the abnormal molecules as well as the aberrant interactions of altered molecules on which cancers depend. Being able to detect such fundamental finger-prints of cancer is key to improved matching between drugs-based treatment and disease. Moreover, imaging-based quantified monitoring of changes in tumor metabolism and its microenvironment could provide real-time non-invasive tool to predict the evolution and progression of primary tumors, as well as the development of tumor metastases.

A recent review-paper: Image-guided interventional therapy for cancer with radiotherapeutic nanoparticles nicely illustrates the role of imaging in treatment guidance through a comprehensive discussion of; Image-guided radiotherapeutic using intravenous nanoparticles for the delivery of localized radiation to solid cancer tumors.

 Graphical abstract

 Abstract

One of the major limitations of current cancer therapy is the inability to deliver tumoricidal agents throughout the entire tumor mass using traditional intravenous administration. Nanoparticles carrying beta-emitting therapeutic radionuclides [DN: radioactive isotops that emits electrons as part of the decay process a list of β-emitting radionuclides used in radiotherapeutic nanoparticle preparation is given in table1 of this paper.) that are delivered using advanced image-guidance have significant potential to improve solid tumor therapy. The use of image-guidance in combination with nanoparticle carriers can improve the delivery of localized radiation to tumors. Nanoparticles labeled with certain beta-emitting radionuclides are intrinsically theranostic agents that can provide information regarding distribution and regional dosimetry within the tumor and the body. Image-guided thermal therapy results in increased uptake of intravenous nanoparticles within tumors, improving therapy. In addition, nanoparticles are ideal carriers for direct intratumoral infusion of beta-emitting radionuclides by convection enhanced delivery, permitting the delivery of localized therapeutic radiation without the requirement of the radionuclide exiting from the nanoparticle. With this approach, very high doses of radiation can be delivered to solid tumors while sparing normal organs. Recent technological developments in image-guidance, convection enhanced delivery and newly developed nanoparticles carrying beta-emitting radionuclides will be reviewed. Examples will be shown describing how this new approach has promise for the treatment of brain, head and neck, and other types of solid tumors.

The challenges this review discusses

  • intravenously administered drugs are inhibited in their intratumoral penetration by high interstitial pressures which prevent diffusion of drugs from the blood circulation into the tumor tissue [1–5].
  • relatively rapid clearance of intravenously administered drugs from the blood circulation by kidneys and liver.
  • drugs that do reach the solid tumor by diffusion are inhomogeneously distributed at the micro-scale – This cannot be overcome by simply administering larger systemic doses as toxicity to normal organs is generally the dose limiting factor.
  • even nanoparticulate drugs have poor penetration from the vascular compartment into the tumor and the nanoparticles that do penetrate are most often heterogeneously distributed

How imaging could mitigate the above mentioned challenges

  • The inclusion of an imaging probe during drug development can aid in determining the clearance kinetics and tissue distribution of the drug non-invasively. Such probe can also be used to determine the likelihood of the drug reaching the tumor and to what extent.

Note: Drugs that have increased accumulation within the targeted site are likely to be more effective as compared with others. In that respect, Nanoparticle-based drugs have an additional advantage over free drugs with their potential to be multifunctional carriers capable of carrying both therapeutic and diagnostic imaging probes (theranostic) in the same nanocarrier. These multifunctional nanoparticles can serve as theranostic agents and facilitate personalized treatment planning.

  • Imaging can also be used for localization of the tumor to improve the placement of a catheter or external device within tumors to cause cell death through thermal ablation or oxidative stress secondary to reactive oxygen species.

See the example of Vintfolide in The Role of Medical Imaging in Personalized Medicine

vinta

Note: Image guided thermal ablation methods include radiofrequency (RF) ablation, microwave ablation or high intensity focused ultrasound (HIFU). Photodynamic therapy methods using external light devices to activate photosensitizing agents can also be used to treat superficial tumors or deeper tumors when used with endoscopic catheters.

  • Quality control during and post treatment

For example: The use of high intensity focused ultrasound (HIFU) combined with nanoparticle therapeutics: HIFU is applied to improve drug delivery and to trigger drug release from nanoparticles. Gas-bubbles are playing the role of the drug’s nano-carrier. These are used both to increase the drug transport into the cell and as ultrasound-imaging contrast material. The ultrasound is also used for processes of drug-release and ablation.

 HIFU

Additional example; Multifunctional nanoparticles for tracking CED (convection enhanced delivery)  distribution within tumors: Nanoparticle that could serve as a carrier not only for the therapeutic radionuclides but simultaneously also for a therapeutic drug and 4 different types of imaging contrast agents including an MRI contrast agent, PET and SPECT nuclear diagnostic imaging agents and optical contrast agents as shown below. The ability to perform multiple types of imaging on the same nanoparticles will allow studies investigating the distribution and retention of nanoparticles initially in vivo using non-invasive imaging and later at the histological level using optical imaging.

 multi

Conclusions

Image-guided radiotherapeutic nanoparticles have significant potential for solid tumor cancer therapy. The current success of this therapy in animals is most likely due to the improved accumulation, retention and dispersion of nanoparticles within solid tumor following image-guided therapies as well as the micro-field of the β-particle which reduces the requirement of perfectly homogeneous tumor coverage. It is also possible that the intratumoral distribution of nanoparticles may benefit from their uptake by intratumoral macrophages although more research is required to determine the importance of this aspect of intratumoral radionuclide nanoparticle therapy. This new approach to cancer therapy is a fertile ground for many new technological developments as well as for new understandings in the basic biology of cancer therapy. The clinical success of this approach will depend on progress in many areas of interdisciplinary research including imaging technology, nanoparticle technology, computer and robot assisted image-guided application of therapies, radiation physics and oncology. Close collaboration of a wide variety of scientists and physicians including chemists, nanotechnologists, drug delivery experts, radiation physicists, robotics and software experts, toxicologists, surgeons, imaging physicians, and oncologists will best facilitate the implementation of this novel approach to the treatment of cancer in the clinical environment. Image-guided nanoparticle therapies including those with β-emission radionuclide nanoparticles have excellent promise to significantly impact clinical cancer therapy and advance the field of drug delivery.

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RAbD Biotech Presents at 1st Pitch Life Sciences-Philadelphia-September 16, 2014

RAbD is a new biotechnology company founded by Fox  Chase Cancer Center investigators Gregory Adams, Ph.D., Matthew Robinson, Ph.D. and Roland Dunbrack, Ph.D. that is focused on the knowledge-based design of antibodies that bind to key functional, often highly conserved and difficult to target epitopes. We are using homology modeling, crystal structures, protein docking and design software and algorithms to drive combinatorial sampling of CDRs to computationally design new antibodies and then express, validate and perform further design in an iterative manner.Brian Smith, Ph.D., MBA is RAbD Biotech’s Business Development Lead.

Contact information for RAbD Biotech:

Website  http://rabdbiotech.com/

LinkedIn

Twitter @RAbDBiotech

The overall goal of RAbD is to

“drug the undruggable”

The company using in silico design methods to design to produce novel antibodies and biomimetics. The company is developing a first in class biomimetic, RaD-003, for the treatment of ovarian cancer.  Ovarian  cancer is one of the most deadly of all women’s cancers, with very low 5 year survival rates.  An expected 22,000 US women a year will be diagnosed and expected 16,000 will die every year.  Cisplatin/paclitaxel therapy is only approved and effective chemotherapy for ovarian cancer yet resistance develops quickly and is common. RaD-003  targets the MISII receptor (Mullerian Inhibiting Substance Type II Receptor), which is expressed on ovarian cancer cells but not on normal ovarian epithelium.

It has been shown that activation of this receptor by the Mullerian Inhibiting Substance (MIS) has antitumor activity in ovarian cancer.

The MISII receptor had been considered undruggable as

  • MIS is too expensive and difficult to produce
  • previous attempts to develop therapeutic antibodies ot MISIIR have proven difficult

Therefore, the company used their computational platform to produce a “first in class” chimeric biomimetic to more effectively target and activate MISIIR.

For  more information about this meeting and the Mid-Atlantic Bioangels and 1st Pitch please see posting on this site

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Good and Bad News Reported for Ovarian Cancer Therapy

Reporter, Curator: Stephen J. Williams, Ph.D.

 

In a recent Fierce Biotech report

FDA review red-flags AstraZeneca’s case for ovarian cancer drug olaparib”,

John Carroll reports on a disappointing ruling by the FDA on AstraZeneca’s PARP1 inhibitor olaparib for maintenance therapy in women with cisplatin refractory ovarian cancer with BRCA mutation.   Early clinical investigations had pointed to efficacy of PARP inhibitors in ovarian tumors carrying the BRCA mutation. The scientific rationale for using PARP1 inhibitors in BRCA1/2 deficiency was quite clear:

  1. DNA damage can result in

1. double strand breaks (DSB)

  1.  DSB can be repaired by efficient homologous recombination (HR) or less efficient non-homologous end joining (NHEJ)

b. BRCA1 involved in RAD51 dependent HR at DSB sites

  1. In BRCA1 deficiency DSB repaired by less efficient NHEJ

 

 

2. single strand breaks, damage (SSB)

  1. PARP1 is activated by DNA damage and poly-ADP ribosylates histones and other proteins marking DNA for SSB repair
  2. SSB repair usually base excision (BER) or sometimes nucleotide excision repair (NER)

B. if PARP inhibited then SSB gets converted to DSB

C. in BRCA1/2 deficient background repair is forced to less efficient NHEJ thereby perpetuating some DNA damage pon exposure to DNA damaging agent

 

A good review explaining the pharmacology behind the rationale of PARP inhibitors in BRCA deficient breast and ovarian cancer is given by Drs. Christina Annunziata and Susan E. Bates in PARP inhibitors in BRCA1/BRCA2 germline mutation carriers with ovarian and breast cancer

(http://f1000.com/prime/reports/b/2/10/) and below a nice figure from their paper:

 

parpbrcadnadamage

 

 

 

 

 

 

 

(from Christina M Annunziata and Susan E Bates. PARP inhibitors in BRCA1/BRCA2 germline mutation carriers with ovarian and breast cancer.  F1000 Biol Reports, 2010; 2:10.)  Creative Commons

Dr. Sudipta Saha’s post BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair discusses how BRCA1 affects the double strand DNA repair process, augments histone modification, as well as affecting expression of DNA repair genes.

Dana Farber’s Dr. Ralph Scully, Ph.D., in Exploiting DNA Repair Targets in Breast Cancer (http://www.dfhcc.harvard.edu/news/news/article/5402/), explains his research investigating why multiple DNA repair pathways may have to be targeted with PARP therapy concurrent with BRCA1 deficiency.

 

However FDA investigators voiced their skepticism of AstraZeneca’s clinical results, namely

  • Small number of patients enrolled
  • BRCA1/2 cohort were identified retrospectively
  • results skewed by false benefit from “underperforming” control arm
  • possible inadvertent selection bias
  • hazard ratio suggesting improvement in progression free survival but higher risk/benefit

The FDA investigators released their report two days before an expert panel would be releasing their own report (reported in the link below from FierceBiotech)

UPDATED: FDA experts spurn AstraZeneca’s pitch for ovarian cancer drug olaparib

in which the expert panel reiterated the findings of the FDA investigators.   The expert panel’s job was to find if there was any clinical benefit for continuing consideration of olaparib, basically stating

“This trial has problems,” noted FDA cancer chief Richard Pazdur during the panel discussion. If investigators had “pristine evidence of a 7-month advantage in PFS, we wouldn’t be here.”

The expert panel was concerned for the above reasons as well as the reported handful of lethal cases of myelodysplastic syndrome and acute myeloid leukemia in the study, although the panel noted these patients had advanced disease before entering the trial, raising the possibility that prior drugs may have triggered their deaths.

 

This was certainly a disappointment as ….

it was at last year’s ASCO (2013) that investigators at Perelman School of Medicine at the University of Pennsylvania and Sheba Medical Center in Tel Hashomer, Israel presented data showing that in 193 cisplatin-refractory ovarian cancer patients carrying a BRCA1/2 mutation, 31% had a partial or complete tumor regression. In addition the study also showed good response in pancreatic and prostate cancer with tolerable side effects.

 

See here for study details: http://www.uphs.upenn.edu/news/News_Releases/2013/05/domchek/

 

As John Carrol from FierceBiotech notes, the decision may spark renewed interest by Pfizer of a bid for AstraZeneca as the potential FDA rejection would certainly dampen AstraZeneca’s future growth and profit plans. Last month AstraZeneca’s CEO made the case to shareholders to reject the Pfizer offer by pointing to AstraZeneca’s potential beefed-up pipeline. AstraZeneca had projected olaparib as a potential $2 billion-a-year seller, although some industry analysts see sales at less than half that amount.

A company spokeswoman said the monotherapy use of olaparib for ovarian cancer assessed by the U.S. expert panel this week was only one element of a broad development program.

 

 

Please see a table of current oncology clinical trials with PARP1 inhibitors

at end of this post

 

However, on the same day, FierceBiotechreports some great news (at least in Europe) on the ovarian cancer front:

 

EU backs Roche’s Avastin for hard-to-treat ovarian cancer

As Arlene Weintraub   of FierceBiotech reports:

EU Committee for Medicinal Products for Human Use (CHMP) handed down a positive ruling on Avastin, recommending that the European Commission approve the drug for use in women with ovarian cancer that’s resistant to platinum-based chemotherapy. It’s the first biologic to receive a positive opinion from the CHMP for this hard-to-treat form of the disease.

Please see here for official press release: CHMP recommends EU approval of Roche’s Avastin for platinum-resistant recurrent ovarian cancer

 

EU had been getting pressure from British doctors to approve Avastin based on clinical trial results although it may be important to note that the EU zone seems to have an ability to recruit more numbers for clinical trials than in US. For instance an EU women’s breast cancer prevention trial had heavy recruitment in what would be considered a short time frame compared to recruitment times for the US.

 

Below is a table on PARP1 inhibitors in current clinical trials (obtained from NewMedicine’s Oncology KnowledgeBase™). nm|OK is a relational knowledgeBASE covering all major aspects of product development in oncolology. The database comprises 6 modules each dedicated in a specific sector within the oncology field.

 

PARP1 Inhibitors Currently in Clinical Trials for Ovarian Cancer

 

Developer and

Drug Name

Development Status & Location
– Indications
AbbVie

Current as of: March 27, 2014

PARP inhibitor: ABT-767

Phase I (begin 5/11, ongoing 2/14) Europe (Netherlands) – solid tumors with BRCA1 or BRCA2 mutations, locally advanced or metastatic • ovarian cancer, advanced or metastatic • fallopian tube cancer, advanced or metastatic • peritoneal cancer, advanced or metastatic
AstraZeneca
Affiliate(s):
· Myriad GeneticsCurrent as of: June 26, 2014Generic Name: Olaparib
Brand Name: Lynparza
Other Designation: AZD2281, KU59436, KU-0059436, NSC 747856
Phase I (begin 7/05, closed 9/08) Europe (Netherlands, UK, Poland); phase II (begin 6/07, closed 2/08, completed 5/09) USA, Australia, Europe (Germany, Spain, Sweden, UK), phase II (begin 7/08, closed 2/09) USA, Australia, Europe (Belgium, Germany, Poland, Spain, UK), Israel, phase II (begin 8/08, closed 12/09, completed 3/13) USA, Australia, Canada, Europe (Belgium, France, Germany, Poland, Romania, Spain, Ukraine, UK), Israel, Russia; phase II (begin 2/10, closed 7/10) USA, Australia, Canada, Europe (Belgium, Czech Republic, Germany, Italy, Netherlands, Spain, UK), Japan, Panama, Peru (combination); MAA (accepted 9/13) EU, NDA (filed 2/14) USA – ovarian cancer, advanced or metastatic, BRCA positive • ovarian cancer, recurrent, platinum sensitive • ovarian cancer, advanced, refractory, BRCA1 or BRCA2-associatedPhase I (begin 5/08, ongoing 5/12) USA; phase II (begin 7/08, closed 10/09) Canada – breast cancer, locally advanced, BRCA1/BRCA2-associated or hereditary metastatic or inoperable • ovarian cancer, locally advanced, BRCA1/BRCA2-associated or hereditary metastatic or inoperable • breast cancer, triple-negative, BRCA-positive • ovarian cancer, high-grade serous and/or undifferentiated, BRCA-positive

Phase I (begin 10/10, ongoing 1/13) USA (combination) – ovarian cancer, inoperable or metastatic, refractory • breast cancer, inoperable or metastatic, refractory

Phase III (begin 8/13) USA, Australia, Brazil, Canada, Europe (France, Italy, Netherlands, Poland, Russia, Spain, UK), Israel, South Korea, phase III (begin 9/13) USA, Australia, Brazil, Canada, Europe (France, Germany, Italy, Netherlands, Poland, Russia, Spain, UK), Israel – ovarian cancer, serous, high grade, BRCA mutated, platinum-sensitive, relapsed, third line, maintenance • ovarian cancer, serous or endometrioid, high grade, BRCA mutated, platinum responsive (PR or CR), maintenance, first line • primary peritoneal cancer, high grade, BRCA mutated, platinum responsive (PR or CR), maintenance • fallopian tube cancer, high grade, BRCA mutated, platinum responsive (PR or

BioMarin Pharmaceutical

Current as of: June 14, 2014

PARP inhibitor:

BMN-673, BMN673, LT-673

Phase I/II (begin 1/11, ongoing 3/14) USA – solid tumors, advanced, recurrent

Phase I (begin 2/13, closed 4/13, completed 5/14) USA – healthy volunteers

Phase I/II (begin 11/13) USA – solid tumors, relapsed or refractory, BRCA mutated, second line

BiPar Sciences

Current as of: April 16, 2009

Parp inhibitor:

BSI-401

Preclin (ongoing 4/09) – solid tumors
Clovis Oncology
Affiliate(s):
· University of Newcastle Upon Tyne
· Cancer Research Campaign Technology
· PfizerCurrent as of: June 21, 2014Generic Name: Rucaparib
Brand Name: Rucapanc
Other Designation: AG140699, AG014699, AG-14,699, AG-14669, AG14699, AG140361, AG-14361, AG-014699, CO-338, PF-01367338
Phase I (begin 03, completed 05) Europe (UK) (combination), phase I (begin 2/10, closed 11/13) Europe (France, UK) (combination) – solid tumors, advanced

Phase II (begin 12/07, closed 10/13) Europe (UK) – breast cancer, advanced or metastatic, in patients carrying BRCA1 or BRCA2 mutations • ovarian cancer, advanced or metastatic, in patients carrying BRCA1 or BRCA2 mutations

Phase I/II (begin 11/11, ongoing 6/14) USA, Europe (UK) – solid tumors, metastatic, with mutated BRCA • breast cancer, metastatic, HEr2 negative, with mutated BRCA

Sanofi

Current as of: June 03, 2013

Generic Name: Iniparib
Brand Name: Tivolza
Other Designation: BSI-201, NSC 746045, SAR240550

Phase I/Ib (begin 3/06, closed 3/10) USA (combination), phase I (begin 7/10, closed 11/10) USA, phase I (begin 9/10, ongoing 2/11) Japan (combination); phase Ib (begin 1/07, ongoing 1/11) USA (combination) – solid tumors, advanced, refractory
Phase II (begin 5/08, closed 1/09) USA – ovarian cancer, advanced, refractory, BRCA-1 or BRCA-2 associated • fallopian tube cancer, advanced, refractory, BRCA-1 or BRCA-2 associated • peritoneal cancer, advanced, refractory, BRCA-1 or BRCA-2 associated
Tesaro
Affiliate(s):
· MerckCurrent as of: May 18, 2014Generic Name: Niraparib
Other Designation: MK-4827, MK4827
Phase I (begin 9/08, closed 2/11) USA, Europe (UK) – solid tumors, locally advanced or metastatic • ovarian cancer, locally advanced or metastatic, BRCA mutant • chronic lymphocytic leukemia (CLL), relapsed or refractory • prolymphocytic leukemia, T cell, relapsed or refractory
Phase Ib (begin 11/10, closed 3/11, terminated 10/12) USA (combination) – solid tumors, locally advanced or metastatic • ovarian cancer, serous, high grade, platinum resistant or refractoryPhase III (begin 5/13, ongoing 5/14) USA – ovarian cancer, platinum-sensitive, high grade serous or BRCA mutant, chemotherapy responsive • fallopian tube cancer • primary peritoneal cancer
Teva Pharmaceutical Industries

Current as of: May 04, 2013

Designation:

CEP-9722

Phase I (begin 5/11, closed 11/12, terminated 10/13) USA, phase I (begin 6/09, closed 7/12, completed 1/12) Europe (France and UK) (combination) – solid tumors, advanced, third line
Phase I (begin 5/11, completed 1/13) Europe (France) (combination) – solid tumors, advanced • mantle cell lymphoma (MCL), advanced

 

 

Summary of Combination Ovarian Cancer Trials with Avastin (current and closed)

 

Indication in Development ovarian cancer, advanced, recurrent, persistent • ovarian cancer, progressive, platinum resistant, second line • fallopian tube cancer, progressive, platinum resistant, second line • primary peritoneal cancer, progressive, platinum resistant, second line
Latest Status Phase II (begin 4/02, closed 8/04) USA, phase II (begin 11/04, closed 10/05) USA; phase III (begin 10/09) Europe (Belgium, Bosnia and Herzegovina, Denmark, Finland, France, Germany, Greece, Italy, Netherlands, Norway, Portugal, Spain, Sweden), Turkey
Clinical History Refer to the Combination Trial Module for trials of Avastin in combination with various chemotherapeutic regimens.According to results from the AURELIA clinical trial (protocol ID: MO22224; 2009-011400-33; NCT00976911), the median PFS in women with progressive platinum resistant ovarian, fallopian tube or primary peritoneal cancer treated with Avastin in combination with chemotherapy, was 6.7 months compared to 3.4 months in those treated with chemotherapy alone for an HR of 0.48 (range =0.38–0.60).. In addition, the objective response rate was 30.9% in women treated with Avastin compared to 12.6% in those on chemotherapy (p=0.001). Certain AE (Grade 2 to 5) that occurred more often in the Avastin arm compared to the chemotherapy alone arm were high blood pressure (20% versus 7%) and an excess of protein in the urine (11% versus 1%). Gastrointestinal perforations and fistulas occurred in 2% of women in the Avastin arm compared to no events in the chemotherapy arm (Pujade-Lauraine E, etal, ASCO12, Abs. LBA5002).A multicenter (n=124), randomized, open label, 2-arm, phase III clinical trial (protocol ID: MO22224; 2009-011400-33; NCT00976911; http://clinicaltrials.gov/ct2/results?term=NCT00976911 ), dubbed AURELIA, was initiated in October 2009, in Europe (Belgium, Bosnia and Herzegovina, Denmark, Finland, France, Germany, Greece, Italy, Netherlands, Norway, Portugal, Spain, and Sweden), and Turkey, to evaluate the efficacy and safety of Avastin added to chemotherapy versus chemotherapy alone in patients with epithelial ovarian, fallopian tube or primary peritoneal cancer with disease progression within 6 months of platinum therapy in the first line setting. The trials primary outcome measure is PFS. Secondary outcome measures include objective response rate, biological PFS interval, OS, QoL, and safety and tolerability. According to the protocol, all patients are treated with standard chemotherapy with IV paclitaxel (80 mg/m²) on days 1, 8, 15 and 22 of each 4-week cycle; or IV topotecan at a dose of 4 mg/m² on days 1, 8 and 15 of each 4-week cycle, or 1.25 mg/kg on days 1-5 of each 3-week cycle; or IV liposomal doxorubicin (40 mg/m²) every 4 weeks. Patients (n=179) randomized to arm 2 of the trial are treated with IV Avastin at a dose of 10 mg/kg twice weekly or 15 mg/kg thrice weekly concomitantly with the chemotherapy choice. Treatment continues until disease progression. Subsequently, patients are treated with the standard of care. Patients in arm 1 (n=182), on chemotherapy only may opt to be treated with IV Avastin (15 mg/kg) three times weekly. The trial was set up in cooperation with the Group d’Investigateurs Nationaux pour l’Etude des Cancers Ovariens (GINECO) and was conducted by the international network of the Gynecologic Cancer Intergroup (GCIG) and the pan-European Network of Gynaecological Oncological Trial Groups (ENGOT), under PI Eric Pujade-Lauraine, MD, Hopitaux Universitaires, Paris Centre, Hôpital Hôtel-Dieu (Paris, France). The trial enrolled 361 patients and was closed as of May 2012..Results were presented from a phase II clinical trial (protocol ID: CDR0000068839; GOG-0170D; NCT00022659) of bevacizumab in patients with persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer that was performed by the Gynecologic Oncology Group to determine the ORR, PFS, and toxicity for this treatment. Patients must have been administered 1-2 prior cytotoxic regimens. Treatment consisted of bevacizumab (15 mg/kg) IV every 3 weeks until disease progression or prohibitive toxicity. Between April 2002 and August 2004, 64 patients were enrolled, of which 2 were excluded for wrong primary and borderline histology and 62 were evaluable (1 previous regimen=23, 2 previous regimens=39). The median disease free interval from completion of primary cytotoxic chemotherapy to first recurrence was 6.5 months. Early results demonstrated that some patients had confirmed objective responses and PFS in some was at least 6 months. Observed Grade 3 or 4 toxicities included allergy (Grade 3=1), cardiovascular (Grade 3=4; Grade 4=1), gastrointestinal (Grade 3=3), hepatic (Grade 3=1), pain (Grade 3=2), and pulmonary (Grade 4=1). As of 11/04, 36 patients were removed from the trial, including 29 for disease progression and 1 for toxicity in 33 cases reported. Preliminary evidence exists for objective responses to bevacizumab (Burger R, et al, ASCO05, Abs. 5009).An open label, single arm, 2-stage, phase II clinical trial (protocol ID: AVF2949g, NCT00097019) of bevacizumab in patients with platinum resistant, advanced (Stage III or IV), ovarian cancer or primary peritoneal cancer for whom subsequent doxorubicin or topotecan therapy also has failed was initiated in November 2004 at multiple locations in the USA to determine the safety and efficacy for this treatment.A multicenter phase II clinical trial was initiated in April 2002 to determine the 6-month PFS of patients with persistent or recurrent ovarian epithelial or primary peritoneal cancer treated with bevacizumab (protocol ID: GOG-0170D, CDR0000068839, NCT00022659). IV bevacizumab is administered over 30-90 minutes on day 1. Treatment is repeated every 21 days in the absence of disease progression or unacceptable toxicity. Patients are followed every 3 months for 2 years, every 6 months for 3 years, and then annually thereafter. A total of 22-60 patients will be accrued within 12-30 months. Robert A. Burger, MD, of Chao Family Comprehensive Cancer Center is Trial Chair.This trial was closed in August 2004.

 

 

Sources

http://www.fiercebiotech.com/story/fda-review-red-flags-astrazenecas-case-ovarian-cancer-drug-olaparib/2014-06-23

 

http://www.fiercebiotech.com/story/fda-experts-spurn-astrazenecas-pitch-ovarian-cancer-drug-olaparib/2014-06-25

 

http://www.fiercepharma.com/story/eu-backs-roches-avastin-hard-treat-ovarian-cancer/2014-06-27

 

In a followup to this original posting A Report From the Institute of Medicine of the National Academies of Sciences, Engineering, and Medicine entitled

Evolving Approaches in Research and Care for Ovarian Cancers

was generated in a ViewPoint piece in JAMA which discussed their Congressional mandated report on the State of the Science in Ovarian Cancer Research, titled

Ovarian Cancers: Evolving Paradigms in Research and Care 

highlights some of the research gaps felt by the committee in the current state of ovarian cancer research including:

  • consideration in research protocols of the multitude of histologic and morphologic subtypes of ovarian cancer, including the feeling of the committee that high grade serous OVCA originates from the distal end of the fallopian tube (espoused by Dr. Doubeau and Dr. Christopher Crum) versus originating from the ovarian surface epithelium
  • a call for expanded screening and prevention research with mutimodal screening including CA125 with secondary transvaginal screen
  • better patient education of the risk/benefit of genetic testing including BRCA1/2 as well as in consideration for PARP inhibitor therapy
  • treatments should be standardized and disseminated including more research in health outcomes and decision support for personalized therapy

This Perspective article can be found here: jvp160038

Some other posts relating to OVARIAN CANCER on this site include

Efficacy of Ovariectomy in Presence of BRCA1 vs BRCA2 and the Risk for Ovarian Cancer

Testing for Multiple Genetic Mutations via NGS for Patients: Very Strong Family History of Breast & Ovarian Cancer, Diagnosed at Young Ages, & Negative on BRCA Test

Ultrasound-based Screening for Ovarian Cancer

Dasatinib in Combination With Other Drugs for Advanced, Recurrent Ovarian Cancer

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

 

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