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Personalized Medicine, Omics, and Health Disparities in Cancer: Can Personalized Medicine Help Reduce the Disparity Problem?
Curator: Stephen J. Williams, PhD
In a Science Perspectives article by Timothy Rebbeck, health disparities, specifically cancer disparities existing in the sub-Saharan African (SSA) nations, highlighting the cancer incidence disparities which exist compared with cancer incidence in high income areas of the world [1]. The sub-Saharan African nations display a much higher incidence of prostate, breast, and cervix cancer and these cancers are predicted to double within the next twenty years, according to IARC[2]. Most importantly,
the histopathologic and demographic features of these tumors differ from those in high-income countries
meaning that the differences seen in incidence may reflect a true health disparity as increases rates in these cancers are not seen in high income countries (HIC).
Most frequent male cancers in SSA include prostate, lung, liver, leukemia, non-Hodgkin’s lymphoma, and Kaposi’s sarcoma (a cancer frequently seen in HIV infected patients [3]). In SSA women, breast and cervical cancer are the most common and these display higher rates than seen in high income countries. In fact, liver cancer is seen in SSA females at twice the rate, and in SSA males almost three times the rate as in high income countries.
Reasons for cancer disparity in SSA
Patients with cancer are often diagnosed at a late stage in SSA countries. This contrasts with patients from high income countries, which have their cancers usually diagnosed at an earlier stage, and with many cancers, like breast[4], ovarian[5, 6], and colon, detecting the tumor in the early stages is critical for a favorable outcome and prognosis[7-10]. In addition, late diagnosis also limits many therapeutic options for the cancer patient and diseases at later stages are much harder to manage, especially with respect to unresponsiveness and/or resistance of many therapies. In addition, treatments have to be performed in low-resource settings in SSA, and availability of clinical lab work and imaging technologies may be limited.
Molecular differences in SSA versus HIC cancers which may account for disparities
Emerging evidence suggests that there are distinct molecular signatures with SSA tumors with respect to histotype and pathology. For example Dr. Rebbeck mentions that Nigerian breast cancers were defined by increased mutational signatures associated with deficiency of the homologous recombination DNA repair pathway, pervasive mutations in the tumor suppressor gene TP53, mutations in GATA binding protein 3 (GATA3), and greater mutational burden, compared with breast tumors from African Americans or Caucasians[11]. However more research will be required to understand the etiology and causal factors related to this molecular distinction in mutational spectra.
It is believed that there is a higher rate of hereditary cancers in SSA. And many SSA cancers exhibit the more aggressive phenotype than in other parts of the world. For example breast tumors in SSA black cases are twice as likely than SSA Caucasian cases to be of the triple negative phenotype, which is generally more aggressive and tougher to detect and treat, as triple negative cancers are HER2 negative and therefore are not a candidate for Herceptin. Also BRCA1/2 mutations are more frequent in black SSA cases than in Caucasian SSA cases [12, 13].
Initiatives to Combat Health Disparities in SSA
Multiple initiatives are being proposed or in action to bring personalized medicine to the sub-Saharan African nations. These include:
H3Africa empowers African researchers to be competitive in genomic sciences, establishes and nurtures effective collaborations among African researchers on the African continent, and generates unique data that could be used to improve both African and global health.
There is currently a global effort to apply genomic science and associated technologies to further the understanding of health and disease in diverse populations. These efforts work to identify individuals and populations who are at risk for developing specific diseases, and to better understand underlying genetic and environmental contributions to that risk. Given the large amount of genetic diversity on the African continent, there exists an enormous opportunity to utilize such approaches to benefit African populations and to inform global health.
The Human Heredity and Health in Africa (H3Africa) consortium facilitates fundamental research into diseases on the African continent while also developing infrastructure, resources, training, and ethical guidelines to support a sustainable African research enterprise – led by African scientists, for the African people. The initiative consists of 51 African projects that include population-based genomic studies of common, non-communicable disorders such as heart and renal disease, as well as communicable diseases such as tuberculosis. These studies are led by African scientists and use genetic, clinical, and epidemiologic methods to identify hereditary and environmental contributions to health and disease. To establish a foundation for African scientists to continue this essential work into the future work, the consortium also supports many crucial capacity building elements, such as: ethical, legal, and social implications research; training and capacity building for bioinformatics; capacity for biobanking; and coordination and networking.
Advancing precision medicine in a way that is equitable and beneficial to society means ensuring that healthcare systems can adopt the most scientifically and technologically appropriate approaches to a more targeted and personalized way of diagnosing and treating disease. In certain instances, countries or institutions may be able to bypass, or “leapfrog”, legacy systems or approaches that prevail in developed country contexts.
The World Economic Forum’s Leapfrogging with Precision Medicine project will develop a set of tools and case studies demonstrating how a precision medicine approach in countries with greenfield policy spaces can potentially transform their healthcare delivery and outcomes. Policies and governance mechanisms that enable leapfrogging will be iterated and scaled up to other projects.
Successes in personalized genomic research in SSA
As Dr. Rebbeck states:
Because of the underlying genetic and genomic relationships between Africans and members of the African diaspora (primarily in North America and Europe), knowledge gained from research in SSA can be used to address health disparities that are prevalent in members of the African diaspora.
For example members of the West African heritage and genomic ancestry has been reported to confer the highest genomic risk for prostate cancer in any worldwide population [14].
Science 03 Jan 2020:
Vol. 367, Issue 6473, pp. 27-28
DOI: 10.1126/science.aay474
Summary/Abstract
Cancer is an increasing global public health burden. This is especially the case in sub-Saharan Africa (SSA); high rates of cancer—particularly of the prostate, breast, and cervix—characterize cancer in most countries in SSA. The number of these cancers in SSA is predicted to more than double in the next 20 years (1). Both the explanations for these increasing rates and the solutions to address this cancer epidemic require SSA-specific data and approaches. The histopathologic and demographic features of these tumors differ from those in high-income countries (HICs). Basic knowledge of the epidemiology, clinical features, and molecular characteristics of cancers in SSA is needed to build prevention and treatment tools that will address the future cancer burden. The distinct distribution and determinants of cancer in SSA provide an opportunity to generate knowledge about cancer risk factors, genomics, and opportunities for prevention and treatment globally, not only in Africa.
Parkin DM, Ferlay J, Jemal A, Borok M, Manraj S, N’Da G, Ogunbiyi F, Liu B, Bray F: Cancer in Sub-Saharan Africa: International Agency for Research on Cancer; 2018.
Chinula L, Moses A, Gopal S: HIV-associated malignancies in sub-Saharan Africa: progress, challenges, and opportunities. Current opinion in HIV and AIDS 2017, 12(1):89-95.
Colditz GA: Epidemiology of breast cancer. Findings from the nurses’ health study. Cancer 1993, 71(4 Suppl):1480-1489.
Hamilton TC, Penault-Llorca F, Dauplat J: [Natural history of ovarian adenocarcinomas: from epidemiology to experimentation]. Contracept Fertil Sex 1998, 26(11):800-804.
Garner EI: Advances in the early detection of ovarian carcinoma. J Reprod Med 2005, 50(6):447-453.
Brockbank EC, Harry V, Kolomainen D, Mukhopadhyay D, Sohaib A, Bridges JE, Nobbenhuis MA, Shepherd JH, Ind TE, Barton DP: Laparoscopic staging for apparent early stage ovarian or fallopian tube cancer. First case series from a UK cancer centre and systematic literature review. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 2013, 39(8):912-917.
Kolligs FT: Diagnostics and Epidemiology of Colorectal Cancer. Visceral medicine 2016, 32(3):158-164.
Rocken C, Neumann U, Ebert MP: [New approaches to early detection, estimation of prognosis and therapy for malignant tumours of the gastrointestinal tract]. Zeitschrift fur Gastroenterologie 2008, 46(2):216-222.
Srivastava S, Verma M, Henson DE: Biomarkers for early detection of colon cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2001, 7(5):1118-1126.
Pitt JJ, Riester M, Zheng Y, Yoshimatsu TF, Sanni A, Oluwasola O, Veloso A, Labrot E, Wang S, Odetunde A et al: Characterization of Nigerian breast cancer reveals prevalent homologous recombination deficiency and aggressive molecular features. Nature communications 2018, 9(1):4181.
Zheng Y, Walsh T, Gulsuner S, Casadei S, Lee MK, Ogundiran TO, Ademola A, Falusi AG, Adebamowo CA, Oluwasola AO et al: Inherited Breast Cancer in Nigerian Women. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2018, 36(28):2820-2825.
Rebbeck TR, Friebel TM, Friedman E, Hamann U, Huo D, Kwong A, Olah E, Olopade OI, Solano AR, Teo SH et al: Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations. Human mutation 2018, 39(5):593-620.
Lachance J, Berens AJ, Hansen MEB, Teng AK, Tishkoff SA, Rebbeck TR: Genetic Hitchhiking and Population Bottlenecks Contribute to Prostate Cancer Disparities in Men of African Descent. Cancer research 2018, 78(9):2432-2443.
Other articles on Cancer Health Disparities and Genomics on this Online Open Access Journal Include:
JP Morgan Healthcare Conference Update: Sage, Mersana, Shutdown Woes and Babies
Published: Jan 10, 2019By Alex Keown
With the J.P. Morgan Healthcare Conference winding down, companies remain busy striking deals and informing investors about pipeline advances. BioSpace snagged some of the interesting news bits to come out of the conference from Wednesday.
SAGE Therapeutics – Following a positive Phase III report that its postpartum depression treatment candidate SAGE-217 hit the mark in its late-stage clinical trial, Sage Therapeutics is eying the potential to have multiple treatment options available for patients. At the start of J.P. Morgan, Sage said that patients treated with SAGE-217 had a statistically significant improvement of 17.8 points in the Hamilton Rating Scale for Depression, compared to 13.6 for placebo. The company plans to seek approval for SAGE-2017, but before that, the FDA is expected to make a decision on Zulresso in March. Zulresso already passed muster from advisory committees in November, and if approved, would be the first drug specifically for postpartum depression. In an interview with the Business Journal, Chief Business Officer Mike Cloonan said the company believes there is room in the market for both medications, particularly since the medications address different patient populations.
Mersana Therapeutics – After a breakup with Takeda Pharmaceutical and the shelving of its lead product, Cambridge, Mass.-based Mersana is making a new path. Even though a partial clinical hold was lifted following the death of a patient the company opted to shelve development of XMT-1522. During a presentation at JPM, CEO Anna Protopapas noted that many other companies are developing therapies that target the HER2 protein, which led to the decision, according to the Boston Business Journal. Protopapas said the HER2 space is highly competitive and now the company will focus on its other asset, XMT-1536, an ADC targeting NaPi2b, an antigen highly expressed in the majority of non-squamous NSCLC and epithelial ovarian cancer. XMT-1536 is currently in Phase 1 clinical trials for NaPi2b-expressing cancers, including ovarian cancer, non-small cell lung cancer and other cancers. Data on XMT-1536 is expected in the first half of 2019.
Novavax – During a JPM presentation, Stan Erck, CEO of Novavax, pointed to the company’s RSV vaccine, which is in late-stage development. The vaccine is being developed for the mother, in order to protect an infant. The mother transfers the antibodies to the infant, which will provide the baby with protection from RSV in its first six months. Erck called the program historic. He said the Phase III program is in its fourth year and the company has vaccinated 4,636 women. He said they are tracking the women and the babies. Researchers call the mothers every week through the first six months of the baby’s life to acquire data. Erck said the company anticipates announcing trial data this quarter. If approved, Erck said the market for the vaccine could be a significant revenue driver.
“You have 3.9 million birth cohorts and we expect 80 percent to 90 percent of those mothers to be vaccinated as a pediatric vaccine and in the U.S. the market rate is somewhere between $750 million and a $1 billion and then double that for worldwide market. So it’s a large market and we will be first to market in this,” Erck said, according to a transcript of the presentation.
Denali Therapeutics – Denali forged a collaboration with Germany-based SIRION Biotech to develop gene therapies for central nervous disorders. The two companies plan to develop adeno-associated virus (AAV) vectors to enable therapeutics to cross the blood-brain barrier for clinical applications in neurodegenerative diseases including Parkinson’s, Alzheimer’s disease, ALS and certain other diseases of the CNS.
AstraZeneca – Pharma giant AstraZeneca reported that in 2019 net prices on average across the portfolio will decrease versus 2018. With a backdrop of intense public and government scrutiny over pricing, Market Access head Rick Suarez said the company is increasing its pricing transparency. Additionally, he said the company is looking at new ways to price drugs, such as value-based reimbursement agreements with payers, Pink Sheet reported.
Amarin Corporation – As the company eyes a potential label expansion approval for its cardiovascular disease treatment Vascepa, Amarin Corporation has been proactively hiring hundreds of sales reps. In the fourth quarter, the company hired 265 new sales reps, giving the company a sales team of more than 400, CEO John Thero said. Thero noted that is a label expansion is granted by the FDA, “revenues will increase at least 50 percent over what we did in the prior year, which would give us revenues of approximate $350 million in 2019.”
Government Woes – As the partial government shutdown in the United States continues into its third week, biotech leaders at JPM raised concern as the FDA’s carryover funds are dwindling. With no new funding coming in, reviews of New Drug Applications won’t be able to continue past February, Pink Sheet said. While reviews are currently ongoing, no New Drug Applications are being accepted by the FDA at this time. With the halt of NDA applications, that has also caused some companies to delay plans for an initial public offering. It’s hard to raise potential investor excitement without the regulatory support of a potential drug approval. During a panel discussion, Jonathan Leff, a partner at Deerfield Management, noted that the ongoing government shutdown is a reminder of how “overwhelmingly dependent the whole industry of biotech and drug development is on government,” Pink Sheet said.
Other posts on the JP Morgan 2019 Healthcare Conference on this Open Access Journal include:
The findings are summarized nicely in the NPR article from Joanne Silberner below but just want to list a few takeaways from the study
Ovarian Cancer, while not the most common cancer in women, is still one of the most deadly malignancies. A major reason for this is the inability to catch the disease in its early, and most treatable stages. Much work is being done on early detection (a few posts on this area from this online journal are given at the end of this post for reference)
The symptoms of ovarian cancer closely mimic symptoms of gastrointestinal distress and disorders and many times these symptoms are overlooked by women as benign, temporary issues and may be mis-self diagnosed. In addition, if mistaken for common gastrointestinal discomfort or gynecologic discomfort (cramping) women may self-medicate with over the counter agents which mask the symptoms of ovarian cancer
certain lessons can be learned from the experiences in other countries regarding access to healthcare and diagnosis. For instance
Looking at the key findings of the study it becomes clear that countries have significant potential to
learn from each other:
• Women in Germany had the shortest time to diagnosis, but much less access to
specialist clinicians that are key to successful treatment.
• Women in the UK have almost universal access to specialists but the lowest
proportion of women diagnosed within a month of visiting a doctor.
• Women in Japan had one of the shortest times to diagnosis, but very little access to
genetic testing, and were least likely to get the emotional support they needed.
• Women in the USA were most likely to wait more than three months before
consulting a doctor about symptoms, but most likely to receive genetic testing.
• Women with ovarian cancer in Hungary were most aware of ovarian cancer before
their diagnosis, but were much less likely to be offered surgery to treat their disease.
In summary it appears there are three key areas needing to be addressed with regard to improving early reporting of symptoms of ovarian cancer
information and awareness of symptoms by BOTH women and their physicians
family risk assessment programs are very important to make women aware of their risks and needs for screening
access to specialist treatment is important in the early diagnosis and treatment of this disease
Historically ovarian cancer was called the “silent killer” because symptoms were not thought to develop until the chance of cure was poor. However, recent studies have shown this term is untrue and that the following symptoms are much more likely to occur in women with ovarian cancer than women in the general population. These symptoms include:
Bloating
Pelvic or abdominal pain
Difficulty eating or feeling full quickly
Urinary symptoms (urgency or frequency)
Women with ovarian cancer report that symptoms are persistent and represent a change from normal for their bodies. The frequency and/or number of such symptoms are key factors in the diagnosis of ovarian cancer. Several studies show that even early stage ovarian cancer can produce these symptoms.
Women who have these symptoms almost daily for more than a few weeks should see their doctor, preferably a gynecologist. Prompt medical evaluation may lead to detection at the earliest possible stage of the disease. Early stage diagnosis is associated with an improved prognosis.
Several other symptoms have been commonly reported by women with ovarian cancer. These symptoms include fatigue, indigestion, back pain, pain with intercourse, constipation and menstrual irregularities. However, these other symptoms are not as useful in identifying ovarian cancer because they are also found in equal frequency in women in the general population who do not have ovarian cancer.
In addition there are serum biomarker tests which have shown useful in the screening for ovarian cancer however these tests have their caveats and not generally suggested for whole population screening due to number of false postitives which may occur (these tests will be discussed in further posts)
A new study of women with ovarian cancer shows that ignorance about the condition is common among patients in all 44 countries surveyed. And that ignorance has a cost. The disease is more treatable, even potentially curable, in its early stages.
The women’s answers also suggested their doctors were ignorant. Many of them reported that diagnosis took a long time and that they weren’t referred to proper specialists.
The study was based on an online survey of 1,531 women who had been diagnosed with the cancer and was conducted by the World Ovarian Cancer Coalition, a nonprofit support group between March and May of this year.
Ovarian cancer is the eighth leading cause of cancer in women, according to the World Health Organization. Nearly 300,000 women will develop it this year. The World Ovarian Cancer Coalition estimates that one in six will die within three months of diagnosis and fewer than half will be alive in five years.
Prior to their diagnosis, two-thirds of the women surveyed either had never heard of ovarian cancer or were familiar with the name but didn’t know anything about the disease.
Other articles related to Ovarian Cancer on this online Open Access Journal Include:
Curation of selected topics and articles on Role of G-Protein Coupled Receptors in Chronic Disease as supplemental information for #TUBiol3373
Curator: Stephen J. Williams, PhD
Below is a series of posts and articles related to the role of G protein coupled receptors (GPCR) in various chronic diseases. This is only a cursory collection and by no means represents the complete extensive literature on pathogenesis related to G protein function or alteration thereof. However it is important to note that, although we think of G protein signaling as rather short lived, quick, their chronic activation may lead to progression of various disease. As to whether disease onset, via GPCR, is a result of sustained signal, loss of desensitization mechanisms, or alterations of transduction systems is an area to be investigated.
Inflammatory and infectious factors are present in diseased airways that interact with G-protein coupled receptors (GPCRs), such as purinergic receptors and bradykinin (BK) receptors, to stimulate phospholipase C [PLC]. This is followed by the activation of inositol 1,4,5-trisphosphate (IP3)-dependent activation of IP3 channel receptors in the ER, which results in channel opening and release of stored Ca2+ into the cytoplasm. When ER Ca2+ stores are depleted a pathway for Ca2+ influx across the plasma membrane is activated. This has been referred to as “capacitative Ca2+ entry”, and “store-operated calcium entry” (3). In the next step PLC mediated Ca2+ i is mobilized as a result of GPCR activation by inflammatory mediators, which triggers cytokine production by Ca2+ i-dependent activation of the transcription factor nuclear factor kB (NF-kB) in airway epithelia.
Larry H. Bernstein, MD, FCAP, Curator discusses findings from a research team at University of California at San Diego (UCSD) which the neuropeptide hormone corticotropin-releasing factor (CRF) as having an important role in the etiology of Alzheimer’s Disease (AD). CRF activates the CRF receptor (a G stimulatory receptor). It was found inhibition of the CRF receptor prevented cognitive impairment in a mouse model of AD. Furthermore researchers at the Flanders Interuniversity Institute for Biotechnology found the loss of a protein called G protein-coupled receptor 3 (GPR3) may lower the amyloid plaque aggregation, resulting in improved cognitive function. Additionally inhibition of several G-protein coupled receptors alter amyloid precursor processing, providing a further mechanism of the role of GPCR in AD (see references in The role of G protein-coupled receptors in the pathology of Alzheimer’s disease by Amantha Thathiah and Bart De Strooper Nature Reviews Feb 2011; 12: 73-87 and read post).
Further curations and references of G proteins and chronic disease can be found at the Open Access journal https://pharmaceuticalintelligence.com using the search terms “GCPR” and “disease” in the Search box in the upper right of the home page.
Almudena’s Story: A Life of Hope, Rejuvenation and Strength
Author: Gail S. Thornton, M.A.
Co-Editor: The VOICES of Patients, HealthCare Providers, Caregivers and Families: Personal Experience with Critical Care and Invasive Medical Procedures
Patient had ovarian clear cell adenocarcinomas (OCCAs) and underwent a complete hysterectomy at age 52. Interview was conducted 15 months’ post-surgery. Earlier in life, patient had thyroid cancer and removal of her thyroid gland and all the lymph nodes in her neck.
Almudena Seeder-Alonso, originally from Madrid, Spain, and now living in Amsterdam, The Netherlands, with her Dutch husband, René, is the eternal optimist, embracing life, reinventing herself, and looking for opportunity in every moment. She is an influential blogger of international relations issues, a career professional in human resources management in both corporate and consulting businesses in Legal, Accounting and Technology, and a lawyer and political scientist with an advanced degree in international relations who is also pursuing a Ph.D. in international relations and diplomacy. And she speaks four languages fluently – Spanish, Dutch, Portuguese and English.
Her story is one of hope, rejuvenation and strength that defines her effervescent personality. One year ago, a routine gynecology exam changed her outlook and perspective on life. She would have never thought that her diagnosis would be ovarian carcinoma of the clear cell, the most aggressive form of cancer.
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Image SOURCE: Photographs courtesy of Almudena Seeder-Alonso. Top Left: Almudena’s parents, María and Angel, and sister, Cristina, and her husband. Top Right: Almudena during chemotherapy last summer (2015). Middle: Almudena attending a wedding in Asturias (northwest Spain – May 2016), Almudena and René in Comporta, Portugal (Summer 2014) and in New York (April 2014). Below left: Almudena in New York (April 2014). Below Right: Almudena’s sisters, María and Cristina with nephew, Jaime (May 2016).
A Small Cyst Turns Into Diagnosis of Ovarian Cancer
In early 2015, Almudena visited her gynecologist in Amsterdam for a regular, yearly appointment.
“I was feeling fine. I had no physical complaints, except for my monthly periods which were heavy. I didn’t think much about it. During my examination, my doctor told me that she found a small cyst on my right ovary and we would just observe it to make sure it was not growing.”
Almudena went back to her gynecologist at the OLVG (Onze Lieve Vrouw Gasthuis https://www.olvg.nl/) in Amsterdam twice over the next month to monitor the cyst, only to find that the cyst was growing slightly. Her gynecologist recommended blood tests, an ultrasound, and a specimen of the cyst to be removed through a laparoscopy, a procedure requiring small incisions made below the navel using specialized tools.
“The pathology report said that the cyst was an aggressive cancer, called ovarian carcinoma of the clear cell. I remember sitting in my doctor’s office once she told me the results of the test, and I got very quiet. I could not believe that this was happening to me. While I was meeting with the doctor, I called my husband to let the doctor inform him about the situation. I was listening to this conversation but from far away. He immediately left his meeting with his client (he is one of two founding partners of SeederdeBoer, a Dutch Consulting & Technology firm), to come home. I left the doctor’s office, went home and cried in my husband’s arms.”
Almudena then called her parents, María and Angel, and her two sisters, María and Cristina who live in Madrid, to tell them the news.
“My Mother was very emotional when she heard about my diagnosis. My Father, who is a quiet man by nature, asked me, ‘How could this be happening to you again?’ I did not have an answer for him.”
Almudena’s father was referring to his daughter’s diagnosis of thyroid cancer in her late 20s.
Diagnosis of Thyroid Cancer As A Young Woman
When Almudena was 27 years old, she was diagnosed with follicular thyroid cancer, a slow-growing, highly treatable type of cancer that forms in follicular cells in the thyroid gland. After a 12-hour surgery to remove the gland through a procedure called a full thyroidectomy, she also needed radiation therapy. Many years later, she is feeling fine and continues to be on thyroid medication for the rest of her life.
“I was not aware at that young age of the scope of the diagnosis, but my life really changed. I was kind of a party animal at the end of the 1980s, and I did not have any amount of energy for that anymore. I needed several months to get back into shape as the scar from the surgery was a large one on the right side of my neck. I could not use my right arm and hand properly for months, even writing was complicated. The worst news came later when I could not get pregnant given the situation that many of my eggs were gone because of radiation. At that moment, egg freezing technology was not as advanced as it is today; it was not normal to freeze eggs for a later time. That was really painful, as I could not become a mother, even after four in vitro fertilization (IVF) cycles.”
According to the National Cancer Institute’s web site, thyroid cancer is a disease in which malignant cancer cells form in the tissues of the thyroid gland. The thyroid is a gland at the base of the throat near the trachea (windpipe). It is shaped like a butterfly, with a right lobe and a left lobe. The isthmus, a thin piece of tissue, connects the two lobes. A healthy thyroid is a little larger than a quarter coin. It usually cannot be felt through the skin. The thyroid uses iodine, a mineral found in some foods and in iodized salt, to help make several hormones. Thyroid hormones control heart rate, body temperature, and how quickly food is changed into energy (metabolism) as well as, it controls the amount of calcium in the blood. http://www.cancer.gov/types/thyroid/patient/thyroid-treatment-pdq
Ovarian Cancer Diagnosis Continues
Almudena then spoke with her physicians in Madrid, as that is where she grew up, to get a second opinion about her ovarian carcinoma diagnosis. The physicians knew her history well and they told her that they did not believe that the follicular thyroid cancer was directly related to the ovarian cancer.
“My local gynecologist in Amsterdam then referred me to a specialist, Dr. J. van der Velden, a gynecologist/oncologist at the Amsterdam Medisch Centrum (AMC), http://www.cgoa.nl/page/view/name/34-wie-we-zijn, one of the top university hospitals in The Netherlands for this surgery and treatment. My husband, René, and I met with Dr. van der Velden, and he told us that my cancer was a fast-spreading condition and I needed to have it removed immediately. He answered our questions, calmed my fears and said he would do everything to help me.
“I have an open attitude towards people so it was easy to create a good connection with the doctors and medical personnel, which I consider very fundamental in such a process. I talked to them about my concerns or doubts and shared my worries about the process that I was going through. I have to say that all of them were wonderful in every aspect!”
Dr. van der Velden explained to Almudena that as clear cell is an aggressive form of ovarian cancer, it would need to be treated that way. One month later, Almudena underwent a procedure called open surgery, rather than laparoscopic surgery, requiring an incision large enough for the doctor to see the cyst and surrounding tissue.
“My incision from the surgery is a constant reminder of the struggle I went through. The cyst, which was 3cm, was a solid mass on my right ovary. It had adhered itself to the ovary and had to be broken to be removed, so some cells spilled out into my reproductive organs, namely, in my uterus and fallopian tubes. During this surgery, which was a complete hysterectomy, the doctor took additional tissue samples of my reproductive organs to be analyzed by pathology. Weeks later, he found no other metastases or extra cancer cells.”
One month later, Almudena’s body was still recovering from the operation. Now, she had to start chemotherapy back at the OLVG.
“The doctor, Dr. W. Terpstra, hematologist/oncologist instructed me that I would be going through six full cycles of chemotherapy, which means full doses of carboplatin & paclitaxel every 21 days. At first, I felt reasonably good, then as each week progressed, I became more and more tired, nauseous, and just feeling terrible. I was not sleeping well and even lost the sensation of my fingers and toes as chemo attacks the nerves, too. Then, I started losing my eyelashes and hair so I shaved my long, flowing hair and wore a scarf wrapped around my head.”
Almudena would report to the hospital for her weekly chemotherapy session, starting at 9am and leaving at 6pm. The medical team would put her in a room with a full-size bed so she can relax during the infusion. Her husband, two sisters and some close friends would take turns accompanying her during this time, as she had a nurturing and caring support network.
“I could not have gone through this condition without my family and friends. It tests your relationships and shows you who your friends really are.”
The chemotherapy affected Almudena’s red blood cell count halfway through the process and she felt weak and tired.
“Anemia is normal during this time, but always being tired made me concentrate and focus on things less. I would watch a movie or read a book through the chemo session, and then I would fall asleep quickly.”
After Almudena finished the complete cycle of chemotherapy infusions, she had a follow-up appointment with her doctor, which included blood work, CT scan, and other diagnostic tests.
“My doctor said the tests results were very good. Now, I see him every three months for a routine visit. That was such a wonderful report to hear.
“During this process I learned to love myself, and pampered myself and my body. I learned to improve in terms of beauty, even in the worst circumstances. I wanted to feel beautiful and attractive for myself and for my close family. After three chemo cycles, I started even to think about how my new hair style would be in the moment that I finished chemo.”
Ovarian Carcinoma Pathophysiology Facts
According to published studies, ovarian clear cell adenocarcinomas (OCCAs) account for less than 5 percent of all ovarian malignancies, and 3.7–12.1 percent of all epithelial ovarian carcinomas. By contrast, early‐stage clear cell ovarian cancer carries a relatively good prognosis. When compared with their serous counterparts, a greater proportion of OCCA tumors present as early‐stage (I–II) tumors, are often associated with a large pelvic mass, which may account for their earlier diagnosis, and rarely occur bilaterally. Very little is known about the pathobiology of OCCA. Between 5 percent and 10 percent of ovarian cancers are associated with endometriotic lesions in which there is a predominance of clear and endometrioid cell subtypes, suggesting that both tumor types may arise in endometriosis. http://www.cancer.gov/types/ovarian/hp/ovarian-epithelial-treatment-pdq
The National Cancer Institute’s web site offers these statistics. In most families affected with the breast and ovarian cancer syndrome or site-specific ovarian cancer, genetic linkage has been found to the BRCA1 locus on chromosome 17q21. BRCA2, also responsible for some instances of inherited ovarian and breast cancer, has been mapped by genetic linkage to chromosome 13q12. The lifetime risk for developing ovarian cancer in patients harboring germline mutations in BRCA1 is substantially increased over that of the general population. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2001101/
Words Of Wisdom
“Throughout this journey, I found myself again in some way and found my strength as well. When it seemed I could not stand it anymore, either physically and mentally, I realized that I could.
“At the beginning of my diagnosis, I asked myself, ‘Why me?’, and I then changed it to, ‘Why not me?’ I discovered that I have the same opportunities as anyone who becomes ill. The important perspective to have is not whining and dwelling on my bad luck. The important thing is to heal, survive, and recover my life, which is very good!
“I learned the real value and importance of things: to differentiate and give real meaning and value to the care and support of my husband, René, who was always there for me, and my parents and sisters, who came to Amsterdam very often during the process. I also made sure that René was well-supported and accompanied by my family. René was feeling terrible for me, but he never showed it — and I learned this fact after I was starting to be back on track.”
Almudena’s Life Today
“At a significant moment in my life during my cancer diagnosis and after a long professional life in many corporate and consulting business in several countries, I decided to re-invent myself and start a new career, this time, in the battle of the opinions. I always liked foreign affairs and diplomacy, so why not share my thoughts and write about current international issues.”
That’s when Almudena started a blog to discuss relevant international political issues with her background specialization in International Relations, International Politics, International Law and Governance.
“I consider myself politically liberal and have been influenced by J.S. Mill and A. Tocqueville’s tradition of thought, as well as their ethical conception of the defense of freedom. This is what I try to capture in my political approach and in this blog. http://almudenas.website/index.php/about-me/
“Regarding my profession, I have already reinvented myself, leaving the corporate life with all that is included regarding life’s standards, and do what really makes me happy, which I´m doing right now. It seems after all, looking back with perspective, I did the right thing.
“I am grateful for my life and never take anything for granted. I am the happiest when I am doing things that please me or give me the utmost satisfaction. I now have balance in my personal and professional life, something that I’ve never had before. My husband, René, likes it too and I have his full support.”
She recently ‘liked’ this saying on LinkedIn, the professional network site, ‘I never lose. I either win or learn,’ which was attributed to Nelson Mandela, the deceased South African anti-apartheid revolutionary, politician and philanthropist.
Almudena’s life continues on a path of balance, richness and thankfulness for the person she is and the many blessings she continues to have along the way.
Editor’s note:
We would like to thank Gabriela Contreras, a global communications consultant and patient advocate, for the tremendous help and support she provided in locating and scheduling time to talk with Almudena Seeder-Alonso.
Almudena Seeder-Alonso provided her permission to publish this interview on August 10, 2016.
Metastatic prostate cancer remains a highly lethal disease with no curative therapeutic options. A significant subset of patients with prostate cancer harbor either germline or somatic mutations in DNA repair enzyme genes such as BRCA1, BRCA2, or ATM. Emerging data suggest that drugs that target poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) enzymes may represent a novel and effective means of treating tumors with these DNA repair defects, including prostate cancers. Here we will review the molecular mechanism of action of PARP inhibitors and discuss how they target tumor cells with faulty DNA repair functions and transcriptional controls. We will review emerging data for the utility of PARP inhibition in the management of metastatic prostate cancer. Finally, we will place PARP inhibitors within the framework of precision medicine–based care of patients with prostate cancer.
Introduction In 2016, prostate cancer is expected to be diagnosed in 180,890 men, and 26,120 will die of metastatic disease.[1] While the majority of localized prostate cancers can be controlled with surgery and/or radiation, metastatic disease remains a lethal disease with no curative options. Moreover, prostate cancer is a heterogeneous disease that can be highly lethal but also slow and indolent, as reflected by a 10-year estimated survival of 17% (S9346 trial, unpublished data). The advent of affordable and efficient techniques for profiling tumors molecularly represents an unprecedented opportunity to better characterize the molecular factors that result in indolent and/or lethal disease and to tailor therapy accordingly. Many clinical trials are already underway to examine whether molecularly targeted therapies can improve outcomes.[2] In this review, we will specifically examine the molecular rationale for one of these targeted approaches, poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibition, in prostate cancer. We will review how PARP inhibitors function as a class, review the molecular features that sensitize cancer cells to this therapy, and discuss the data supporting its potential for patients with prostate cancer. We will then outline a strategy for further development of PARP inhibitors in the prostate cancer field. Metastatic prostate cancer is typically categorized as hormone-sensitive prostate cancer (HSPC), which responds to androgen ablation, or castration-resistant prostate cancer (CRPC), which develops resistance to gonadal suppression. Although bilateral orchiectomy is the historic gold-standard treatment for metastatic HSPC, gonadal suppression is currently accomplished with gonadotropin-releasing hormone agonists or antagonists with or without androgen receptor blockade. This approach remains the cornerstone of therapy for men with metastatic HSPC.[3] Emerging data from large phase III trials (CHAARTED and Systemic Therapy in Advancing or Metastatic Prostate Cancer: Evaluation of Drug Efficacy [STAMPEDE]) have also revealed a large survival benefit for the combination of docetaxel and androgen deprivation in metastatic HSPC.[4,5]
Despite these initially effective treatments, the vast majority of men with metastatic HSPC will progress to CRPC, which is the lethal stage of the disease. For these patients, several additional therapies provide benefit by further suppression of androgen signaling (enzalutamide, abiraterone), disruption of the cell cycle in replicating cells (docetaxel, cabazitaxel), targeting of bone metastases (radium-223), or activation of antitumor immunologic response (sipuleucel-T).[6] While these therapies have undoubtedly extended the median survival of patients with metastatic CRPC, their impact on survival is modest and they clearly do not work for all men. In addition, we lack validated genomic markers that would allow better selection of patients for these therapies. Therefore, a better approach that leverages the individual and unique aspects of a patient’s cancer and utilizes therapy based on these factors may allow us to improve patient outcomes.
The development of high-throughput sequencing technology has made it feasible to comprehensively analyze the genetic mutations and gene expression changes in individual prostate cancers with a high degree of resolution in real time. Many institutions now routinely perform these analyses in the hope that they might uncover molecular features that predict response to certain therapies or provide guidance for clinical trial selection.[7] This approach, colloquially termed “precision” medicine, offers the potential promise of providing the right therapy for the right patient at the right time. In the context of prostate cancer, it means molecularly characterizing a tumor and then offering patients drugs that may specifically promote tumor lethality based on these molecular features. The limitation of this approach is that it requires that the target be truly biologically relevant and that there are drugs that can effectively target these molecular changes. The discovery of both somatic and germline DNA repair deficiencies in prostate cancer, together with the development of PARP inhibitors that can kill cancer cells with these defects, is a potent example of targeting therapy to molecularly defined tumor subtypes. While much early work validating this approach has occurred in breast and ovarian cancer populations, emerging data suggest that PARP inhibition is a potentially important strategy for managing a significant subset of prostate cancer patients.
PARP Inhibition: Targeting DNA Repair Deficiency
Molecular mechanism
PARP1 catalyzes the addition of poly(ADP)-ribose (PAR) groups to target proteins in a process termed PARylation.[8] PARP1 is part of a superfamily of proteins that consists of 18 members (including the related tankyrase enzymes), which have many functions within normal and cancer cells. PARP1, the founding member of this family, is responsible for the majority of PARylation of protein targets within cells. It is primarily present in the nucleus in association with chromatin, where it participates in DNA repair and regulation of gene expression by modulating protein localization and activity.[9]
DNA damage occurs continuously in all living cells as a result of oxidative damage or DNA replicative stress.[10] When DNA damage occurs on one strand of the DNA double helix, a single-strand break (SSB) results, but if two SSBs occur in close proximity and on opposite strands, the result is a double-strand break (DSB) and discontinuity of the chromosome (Figures 1 and 2). Even a single DSB is lethal to a human cell if unrepaired because of the risk of large-scale loss of genetic information.
PARP1 plays a critical role in restoration of genomic integrity by facilitating efficient repair of DNA SSBs and DSBs. PARP1 senses DNA damage by binding to the site of SSBs and DSBs and inducing auto-PARylation, which in turn promotes recruitment of DNA repair factors (such as DNA ligase III, polymerase β, and x-ray repair cross-complementing protein 1[XRCC1]).[11] Loss of PARP1 function by means of pharmacologic or genetic mechanisms results in impaired SSB repair and, following initiation of DNA replication, creation of a DNA DSB (see Figure 1). PARP may also play an important role in DSB repair and is known to recruit the MRE11-RAD50-NBS1 complex and to promote PARylation of BRCA1, factors required for the homologous recombination (HR) pathway of DNA DSB repair. Therefore, pharmacologic inhibition of PARP1/2 in DNA repair–defective (DRD) cells that lack efficient HR repair capabilities (such as those harboring BRCA1, BRCA2, or ATM mutations) results in failure to resolve SSBs, which are then converted to DSBs that promote cellular death.
The activity of PARP1 is not limited to DNA damage response. PARP1 is also known to regulate gene expression by modulation of transcription factor activity and regulation of chromatin.[12] PARP1 binds to RNA polymerase II, regulating gene expression, and may also affect tumor suppressor and oncogenic gene expression. PARP1 can also modulate hormone-dependent gene transcription from hormone-responsive nuclear receptors, such as estrogen receptors α and β, progesterone receptor, and androgen receptor.[9]
Furthermore, PARP1 can modulate the transcriptional activity of ETS transcription factors, which suggests that pharmacologic targeting of PARP1 may be useful in TMPRSS2:ERG fusion–positive prostate cancer cells (~50% of prostate cancers).[13] PARP1 physically interacts with the TMPRSS2:ERG gene fusion and the DNA–protein kinase complex, and these interactions are required for ERG-related gene transcription. Interestingly, PARP inhibition with olaparib inhibited prostate cancer xenograft growth if tumors harbored a TMPRSS2:ERG fusion, which suggests that PARP might represent a therapeutic option for prostate cancer patients withTMPRSS2:ERG fusions.[13] This concept is being evaluated in a recently completed clinical trial (National Cancer Institute [NCI] 9012).
PARP inhibitors
Given the biologic importance of PARP1 in the context of cancer, several pharmacologic agents that target this enzyme are currently under development (Table). Most PARP inhibitors mimic the NAD+ substrate of PARP1, competitively bind to the catalytic domain, and inhibit PAR synthesis.[14] PARP inhibitors require the expression of PARP1 and PARP2, and cells that lack expression of both genes are not sensitive to these agents. PARP inhibitors all appear to block catalytic activity and PAR synthesis in a roughly equivalent manner but may show differential ability to trap PARP1/2 at the site of DNA damage (niraparib > olaparib > veliparib), an event that blocks repair and promotes cellular lethality.[15,16] Whether these effects observed in vitro translate into clinically meaningful differences in efficacy is less clear. Furthermore, it is also now clear that the putative PARP inhibitor iniparib may not promote cytotoxicity via PARP inhibition. Several initial studies focused on iniparib, but when phase III trials failed to demonstrate the efficacy of this compound, additional mechanistic work demonstrated that iniparib may not truly be an effective PARP inhibitor.[17,18] These data illustrate the necessity of careful mechanistic characterization of any targeted agent prior to large-scale and expensive studies.
Germline DNA repair deficiency
Inherited defects in DNA repair pathways result in increased susceptibility to the development of malignancy.[19] Defects in mismatch repair proteins promote the development of tumors, including colon and uterine,[20] whereas inherited inactivating mutations in BRCA1 and BRCA2, which are required for efficient HR-based DNA DSB repair, significantly increase the risk of breast, ovarian, prostate, and other cancers.[21] Patients with these tumor types typically demonstrate homozygous inactivation of these genes, the first event occurring in the germline, with subsequent clonal somatic inactivation of the remaining allele.[21] These events presumably occur early in tumorigenesis and, by loss of robust DNA DSB repair, induce genomic instability, which causes loss of tumor suppressors, activation of oncogenes, and acceleration of tumorigenesis.
A germline mutation in BRCA1 or BRCA2 increases the risk of prostate cancer and thus may be found in 2% to 5% of prostate cancers.[22,23] The relative risk of development of prostate cancer for men ≤ age 65 with BRCA1 mutations is 1.8, but BRCA2 mutations in particular seem to increase the risk of prostate cancer formation by age 65 by about 8.6-fold. Mutations of BRCA1, BRCA2, and ATM (and perhaps other DNA repair genes) may also play a role in progression to the lethal castration-resistant state.[22,24-26] The frequency of BRCA2 germline mutations in prostate cancer alone may be as high as 2%.[22] Therefore, the development of therapies to target DNA repair is likely to benefit a relatively large and relatively young population.
Somatic DNA repair deficiency
In addition to germline defects, tumors can acquire defective DNA repair processes through somatic loss of DNA damage response genes, and these somatic mutations can also confer sensitivity to PARP inhibition.[27] This has led to the concept of “BRCAness,” which refers to somatically acquired defects in HR that, as a group, could predict tumor response to PARP inhibitors and cisplatin.[21] Somatic alterations can include either acquired mutations or epigenetic events that silence genes such as ATM; ATR; BRCA1 or –2; CHEK1 or -2; FANCA, -C, -D2, -E, -F; PALB2; MRE11 complex; or RAD51, which prevent efficient HR repair of DNA DSBs.
It is likely that a substantial proportion of men with prostate cancer may demonstrate aspects of BRCAness that could predict sensitivity to PARP inhibitors. Beltran et al performed targeted next-generation sequencing of tumors from men with advanced prostate cancer and found that 12% demonstrated BRCA2 loss and that 8% harbored ATM loss.[28] Furthermore, up to 19.3% of CRPCs demonstrate aberrations in BRCA1, BRCA2, or ATM; these events become more frequent as the disease progresses from hormone-sensitive to castration-resistant.[29] Together these data suggest that BRCAness is a reasonably frequent event in patients with advanced prostate cancer, which makes PARP inhibition an attractive target in this disease.
Synthetic lethality
The concept of promoting the killing of cancer cells by simultaneously blocking SSB repair using PARP inhibition in cells that lack efficient DSB repair is called “synthetic lethality.” In this scenario, tumor cells may harbor either germline or somatically acquired homozygous inactivation of HR. Germline defects (when present) typically affect only one allele in normal cells, and therefore normal tissues retain HR function. This difference between the DNA repair capacity of normal and cancer cells can be leveraged to produce selective cell killing of tumor cells by PARP inhibitors. Treatment of patients with PARP inhibitors will then block normal SSB repair in all cells, and these SSBs are subsequently converted to DSBs by DNA replication. In normal cells, HR restores the genome and allows survival, but in DRD cancer cells, DSBs persist, inducing cellular death selectively in the tumor cell population (see Figure 2).
Ample data indicate that PARP inhibitors possess antitumor activity within diverse patient populations, particularly those with BRCA1 or BRCA2 mutations.[14] One of the first studies to validate the concept of clinical benefit in patients with BRCA mutations was a phase I trial that looked at pharmacokinetic and pharmacodynamic aspects of olaparib treatment.[24] In this study, 60 patients with solid tumors were treated with various doses of olaparib (10 mg daily to 600 mg twice daily) to determine maximum tolerated dose (MTD). The study population was intentionally enriched for BRCA mutation carriers, and 22 patients of the cohort harbored BRCA1 or BRCA2 mutations. Objective tumor activity was observed in the mutation carrier population in patients with breast, ovarian, and prostate cancers. Three patients with advanced prostate cancer were included in this study cohort; the one with a BRCA2 mutation had a greater than 50% response in prostate-specific antigen (PSA) level, resolution of bone metastases, and an extended treatment course. This study suggested that there was a benefit of olaparib therapy in BRCA mutation carriers and the potential for benefit in prostate cancer patients. Further validation of olaparib efficacy in patients with BRCA mutations came from parallel proof-of-concept studies demonstrating the activity of this agent in women with breast and ovarian cancers and BRCA1 or BRCA2 mutations.[30,31] These data ultimately led to US Food and Drug Administration (FDA) approval of olaparib for women with a BRCA mutation and metastatic ovarian cancer after chemotherapy. Additional data that demonstrate a similar spectrum of activity are available for other PARP inhibitors. Phase I data on the safety and pharmacodynamics of single-agent veliparib have been reported as an abstract,[32] and additional studies of veliparib in combination with mitomycin,[33] irinotecan,[34] and other agents have been reported.[35] VanderWeele et al published a case report of a patient with metastatic CRPC and BRCA2 mutation who had a sustained complete response to veliparib and carboplatin/gemcitabine.[36] It seems likely that many of the available PARP inhibitors may have overlapping activities, and further data will be needed to clarify which agent to use in which tumor type and the relative toxicities of each agent.
emozolomide and veliparib in metastatic CRPC
Compelling data implicate PARP1 in the mediation of DNA repair responses to alkylating agents,[37] cellular survival in BRCA-deficient cells,[24,38] and androgen receptor–mediated prostate cancer cellular proliferation.[9,39] Furthermore, data suggest that prostate cancers that harbor the TMPRSS2:ERG fusion (present in up to 50% of prostate cancers) may be more sensitive to PARP inhibition.[13] Therefore, Hussain et al carried out a single-arm pilot study to assess the safety and efficacy of veliparib with the alkylator temozolomide (TMZ) in patients with metastatic CRPC following docetaxel therapy.[40] In this study, patients with a PSA level of ≥ 2 ng/mL were treated with veliparib, 40 mg twice daily, on days 1 to 7 and TMZ, 150 to 200 mg/m2, on days 1 to 5 on a 28-day cycle, based on tolerance data from a phase I study (ClinicalTrials.gov identifier: NCT00526617). The primary endpoint was PSA response rate (30% decline). Of the 25 patients who were evaluable for response, 2 had a confirmed response, 13 had stable PSA, and 10 had progression. The most frequent toxicities were thrombocytopenia, anemia, fatigue, neutropenia, nausea, and constipation. The investigators did assess frequency of TMPRSS2:ERG fusion but found it in only one of eight evaluable patients. Although this patient had stable disease, no conclusions could be drawn regarding the contribution of the fusion product to veliparib sensitivity. Overall, while the combination was considered tolerable, it had only modest activity. No preselection was done in the study, and because BRCAness exists in 20% of patients, it is perhaps not surprising that activity was modest. The lower dose of PARP inhibitor and the lack of established benefit for TMZ may also have contributed to less than robust clinical activity for this combination. Given the emerging molecular data, it seems that future studies will be more likely to identify activity if done in preselected patient populations.
TOPARP
The Trial of PARP Inhibition in Prostate Cancer (TOPARP-A) sought to determine whether patients with prostate cancers with molecularly identified defects in DNA repair benefited from full-dose olaparib therapy.[25] In this phase II study, 50 men with CRPC underwent biopsy of metastatic disease and targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase chain reaction. The primary endpoint was response rate (either objective response or reduction of 50% in PSA level or reduction in circulating tumor cells). All had previously received docetaxel, and most had been treated with abiraterone or enzalutamide (98%) and cabazitaxel (58%). Patients were grouped according to the presence or absence of a homozygous deletion of or deleterious mutation in DNA damage response genes, which predict sensitivity to PARP inhibition. Overall, 16 of 49 evaluable patients (33%) were biomarker positive (indicative of homozygous deleterious changes in BRCA1/2, ATM, Fanconi anemia genes, or CHEK2). Of these, five patients had germline and somatic events (three patients with germline BRCA2 and three patients with germline ATM deletions or mutations). Of the 16 patients with deleterious changes in DNA repair genes, 14 (88%) responded to olaparib. The median overall survival for patients with biomarker-positive DRD tumors who received olaparib was 13.8 months, compared with 7.5 months for those with biomarker-negative tumors (P = .05). Interestingly, two biomarker-negative patients also met criteria for response to olaparib. Although one was a longer-term responder still on therapy at the time of publication, this particular patient did harbor monoallelic deletions of both BRCA2 and PALB2 that did not meet criteria for the prespecified biomarker-positive category but that may have contributed to tumor sensitivity. Toxicity was as expected, with patients displaying grade 3 or 4 anemia (10/50), fatigue (6/50), leukopenia (3/50), thrombocytopenia (2/50), and neutropenia (2/50). These results illustrate the feasibility of using molecular profiling to identify prostate cancers that display molecular features suggestive of sensitivity to PARP inhibition (BRCAness).
NCI 9012
ETS gene fusions—which result from gene rearrangement and juxtaposition of an androgen-responsive gene, such as TMPRSS2, to an ETS transcription factor gene, such as ERG or ETV1—occur in 50% to 60% of prostate cancers.[41,42] ETS transcription factors may also physically interact with PARP1, and PARP1 activity may be required for ETS-mediated invasion, transcription, and metastasis.[13] Androgen receptor–mediated transcription may also promote DNA DSBs and requires PARP activity for efficient repair.[43-45] Therefore, therapeutic targeting of androgen receptor signaling and PARP1 activity using abiraterone and veliparib is an attractive strategy in the management of metastatic prostate cancer.
A randomized phase II clinical trial in patients with metastatic CRPC was recently completed; it examined whether ETS fusion is a biomarker of response to abiraterone or abiraterone plus veliparib. In this study, 148 patients with metastatic CRPC underwent biopsy followed by assessment of ETS fusion status and then random assignment to either abiraterone alone or abiraterone plus veliparib. The primary endpoint was confirmed PSA response in patients receiving either abiraterone alone or combination therapy, stratified by ETS status. Secondary endpoints included safety, objective response rate, progression-free survival, and whether DNA repair gene deficiency (homozygous deletions of or deleterious mutations in: BRCA1, BRCA2, ATM, FANCA, PALB2, RAD51B, RAD51C) predicts response. This trial has now completed enrollment, and preliminary results will be presented at the American Society of Clinical Oncology 2016 Annual Meeting. Although final results are pending, the study does illustrate the feasibility of a large-scale metastatic tissue–based, biomarker-driven trial involving PARP inhibition in patients with metastatic CRPC. This study will also begin to ascertain the role of ETS fusions in determining response to PARP inhibitor therapy and will further explore the contribution of DRD to patient outcomes in those treated with standard therapy (abiraterone arm) and those treated with PARP inhibition (abiraterone plus veliparib arm).
Future studies
Given the data from the studies discussed previously and the enthusiasm for molecularly targeted trials in oncology, there is interest in further testing of PARP inhibition in prostate cancer patients. Multiple trials have recently been completed, are actively enrolling, or are nearing activation within this space (see Table, ClinicalTrials.gov).
Olaparib. Olaparib is the agent that is farthest along in clinical development and has an FDA indication in ovarian cancer. Olaparib also has the most active or pending studies in prostate cancer patients. TOPARP continues to enroll patients with metastatic CRPC, with a target accrual of 98 patients (ClinicalTrials.gov identifier: NCT01682772). There is a randomized double-blind, placebo-controlled phase II study of abiraterone plus olaparib or placebo for patients with metastatic CRPC who received prior docetaxel therapy (ClinicalTrials.gov identifier: NCT01972217). This trial, which is similar to the NCI 9012 study, has completed enrollment, but results are pending. Another trial is examining the biologic effect of olaparib on prostate cancer specimens when given alone or in combination with degarelix prior to prostatectomy (ClinicalTrials.gov identifier: NCT02324998). Furthermore, there is an open-label phase II study to assess the efficacy and safety of olaparib in patients with BRCA1 or BRCA2 mutations (regardless of tumor type), which is ongoing but no longer enrolling patients (ClinicalTrials.gov identifier: NCT01078662).
Veliparib. NCI 9012 (discussed previously) will help determine whether veliparib has potential therapeutic activity in metastatic CRPC and may identify molecularly determined subsets of disease (ie, ETS fusion–positive, DRD-positive) that might be expected to show the most benefit. The results of this study may help determine whether additional studies of this agent within the prostate cancer space are warranted.
Niraparib. The Hoosier Cancer Research Network has a planned phase I study of the combination of enzalutamide and niraparib for patients with metastatic CRPC (ClinicalTrials.gov identifier: NCT02500901), which has not yet begun enrollment. The primary endpoint of this study will be determination of MTD and dose-limiting toxicity.
Talazoparib. Although no prostate cancer–specific trials using other PARP inhibitors are currently active, several trials for molecularly targeted patient populations or phase I trials for toxicity assessment in combination with chemotherapy are ongoing; these provide some information on prostate cancer populations, depending on the types of solid tumors enrolled. There is a phase I trial of talazoparib in combination with carboplatin and paclitaxel (ClinicalTrials.gov identifier: NCT02317874) and another for patients with solid tumors and hepatic and renal dysfunction (ClinicalTrials.gov identifier: NCT02567396).
Precision Targeting of the PARP Pathway in Prostate Cancer
PARP inhibitors are a promising therapeutic option for men with prostate cancer. There is good evidence that men with either germline or somatic mutations in DNA repair pathways can derive therapeutic benefit from inhibition of PARP1/2, which blocks repair of SSB, driving persistent DSBs that lead to cancer cell lethality. Preclinical data also suggest that PARP inhibition may produce benefits by targeting chromatin and gene transcription, which implies that clinical benefits may extend beyond patients with DRD tumors.[12] To continue to develop PARP inhibitors within the prostate cancer field, we will need to develop and refine a set of biomarkers for use in selecting the right patient populations for these agents and then incorporate these biomarkers into prospective studies. As part of a precision therapy strategy, PARP inhibitors will likely play an important role in the management of prostate cancer in the near future.
It is now feasible to comprehensively profile the mutational, epigenetic, and gene expression changes in men with prostate cancer, and we are beginning to use this information to guide treatment choices.[7] Unfortunately, the functional relevance of many of the molecular features uncovered in these profiles is not completely understood. DNA repair processes are complex and require many genes for efficient repair of various types of DNA damage. Most past and ongoing studies focused on patients with specific molecular features, such as BRCA1, BRCA2, ATM, FANCA, PALB2, RAD51B, and RAD51C mutations. While mutations of these genes are likely to affect sensitivity to PARP inhibitors, mutations in other DNA repair or transcription factor genes may as well, and identification of those genes could expand the patient population that could benefit from therapy. Determination of whether other genes are susceptible to PARP inhibitor therapy will require robust preclinical models with a wide selection of genetic changes that reflect human disease; such models can be used to determine whether additional mutations and epigenetic or gene expression changes also result in PARP inhibitor sensitivity. Given the potential infrequency of many of the individual mutations that might sensitize to PARP inhibitors, large-scale registries that catalog mutations and their responsiveness to therapies may be needed.
As we define the molecular features that suggest sensitivity to PARP inhibition, the challenge will then become understanding the best strategy for incorporating these targeted agents into our standard treatment algorithms. In the context of prostate cancer, PARP inhibitors could be considered in high-risk patient populations in an adjuvant manner, before or with androgen deprivation therapy (ADT) in patients with newly metastatic disease, or in the setting of castration-resistant disease before or after the many other therapeutic options. To date, most trials in the prostate cancer space have been in the castration-resistant setting, perhaps because mutations in DNA damage genes may become more common as the disease progresses.[25] Nonetheless, there is no reason to assume that patients who harbor mutations may not benefit earlier in the disease course. Adjuvant use of PARP inhibitors in those with high-risk or micrometastatic disease could conceivably render patients disease free. Similarly, the combination of ADT and PARP inhibitors in early metastatic disease may provoke prolonged progression-free intervals similar to the situation with early docetaxel therapy but with less toxicity.[4,5] In the context of castration-resistant disease, it is reasonable to hypothesize that the combination of PARP inhibitors with hormonal agents such as abiraterone or enzalutamide or with chemotherapies might act synergistically to promote disease control.
The trials to examine these questions may be more challenging to design and execute because patients with sensitizing molecular changes represent a limited subset of total patients with prostate cancer. This means that in order to identify the subset that will benefit, many will need to be screened.[25] Because most molecular analyses are done using biopsy tissue, screening and cost may be challenging factors. In addition, the natural history of patients with DNA damage pathway mutations may also be distinct from those without such mutations. It is conceivable that mutations in DNA damage response genes may modulate patient response to standard hormonal agents, chemotherapy, or radium because all three of these therapeutic modalities have the potential to induce DNA damage in prostate cancer cells. Given these caveats, it will be essential to design an efficient precision medicine clinical trial pipeline that can rapidly molecularly profile patient tumors, assign to a therapeutic intervention, and then assess the complex resulting data and analyze results according to molecular categories.
PARP inhibitors have the potential to be a promising addition to the therapeutic arsenal used to treat prostate cancer and other solid tumors that harbor the appropriate molecular features. The transition from a standard, one-size-fits-all approach to a targeted, precision medicine strategy in which an individual prostate cancer patient’s tumor biology will guide choice of therapy will require careful planning and thought. The inclusion of PARP-targeted therapies before, after, with, or in place of standard hormonal therapies and chemotherapies will need to be defined so as to maximize antitumor effect and patient survival. Hopefully, application of these novel combinations in those most likely to benefit will ultimately lead to longer and better lives for patients with prostate cancer.
Financial Disclosure:Dr. Hussain is the principal investigator for a clinical trial of veliparib through the Cancer Therapy Evaluation Program (for AbbVie), and is collaborating on a clinical trial of olaparib for AstraZeneca.
David B. Solit, MD Philip W. Kantoff, MD Memorial Sloan Kettering Cancer Center, New York, New York
How an Ovarian Cancer Drug Came to Have ‘Breakthrough Therapy Designation’ for Prostate Cancer
With the emergence of precision medicine, clinicians can now take advantage of high-throughput tumor sequencing to identify driver mutations in individuals with cancer, with the goal of matching these with effective therapies. Since driver mutations can be shared across cancer types, precision medicine has also challenged the notion that cancer types, as defined by site of origin, are completely separate entities. One such example is the use of vemurafenib in multiple BRAF V600–mutant cancers. Another example is that of poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitors and prostate cancer. It is now recognized that DNA repair abnormalities, including and most notably BRCA2 mutations, are found frequently in the germline and as somatic mutations in the tumors in men with metastatic prostate cancer. Moreover, recent studies have demonstrated promising activity for olaparib—a drug approved for use in BRCA-mutated ovarian cancer—in men with castration-resistant disease and germline or somatic DNA repair abnormalities. This has led the US Food and Drug Administration to confer “breakthrough therapy designation” on olaparib, based on the strong belief that the drug will ultimately be approved for this indication.
What Questions Should Future Research on PARP Inhibitors for Prostate Cancer Focus on?
Many questions still remain unanswered. These include:
1) Given the pleiotropic effects of PARP inhibitors, which activities are the most critical and which PARP inhibitors are best for each disease/mutation scenario?
2) Have we identified the full gamut of DNA repair abnormalities that might respond to PARP inhibition?
3) Can we extend the spectrum of patients eligible for PARP inhibitors to those who are homologous recombination–proficient, by combining PARP inhibitors with therapies such as alkylating agents or antiangiogenic agents like cediranib?
4) Can we identify patients early on in their disease course in whom PARP inhibition may contribute to a curative strategy?
Cervical cancer (CC) remains a major health problem worldwide. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutics in ovarian cancer. We explored the preclinical in vitro and in vivo activity of olaparib against multiple primary whole exome sequenced (WES) CC cells lines and xenografts.
Methods.
Olaparib cell-cycle, apoptosis, homologous-recombination-deficiency (HRD), PARP trapping and cytotoxicity activity was evaluated against 9 primary CC cell lines in vitro. PARP and PAR expression were analyzed by western blot assays. Finally, olaparib in vivo antitumor activity was tested against CC xenografts.
Results.
While none of the cell lines demonstrated HRD, three out of 9 (33.3%) primary CC cell lines showed strong PARylation activity and demonstrated high sensitivity to olaparib in vitro treatment (cutoff IC50 values < 2μM, p=0.0012). Olaparib suppressed CC cell growth through cell cycle arrest in the G2/M phase and caused apoptosis (p<0.0001). Olaparib activity in CC involved both PARP enzyme inhibition and trapping. In vivo, olaparib significantly impaired CC xenografts tumor growth (p=0.0017) and increased overall animal survival (p=0.008).
Conclusions.
A subset of CC primary cell lines is highly responsive to olaparib treatment in vitro and in vivo. High level of PARylation correlated with olaparib preclinical activity and may represent a useful biomarker for the identification of CC patients benefitting the most from PARPi.
Despite the implementation of prophylactic vaccination strategies against Human Papillomavirus (HPV) infection and advances in chemoradiation and immunotherapy, cervical cancer (CC) remains a major health problem in the United States with 13,240 new cases and 4,170 related deaths in 2018 [1]. Chemoradiation represents the standard of care for patients with locally advanced disease not suitable for curative surgery [2] while the usual treatment for recurrent/metastatic CC is a combination of paclitaxel and cisplatin or paclitaxel, cisplatin and bevacizumab. These chemotherapy treatments, although not curative, result in median survival times of approximately one to 1.5 years [3–5]. Once patients progress after this initial therapy for recurrent or metastatic disease, options are limited (there are no FDA approved or NCCN level 1 or 2A therapies available). Identification of novel, effective therapies for CC patients with disease resistant to standard treatment modalities remains an unmet medical need.
In recent years, Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutic agents for ovarian cancer associated with defects in homologous recombination DNA repair (HRR) system [6–10]. PARP1 is one of the most abundant proteins among several members of the PARP family and multiple studies implicated PARP1 as having pleiotropic cellular functions, such as maintenance of genomic integrity, DNA repair and regulation of apoptotic and survival balance in cells [11–14]. Furthermore, the enzyme is involved in the PARylation of nuclear proteins (i.e., the post-translational modification process by which polymers of ADP-ribose (poly(adenosine diphosphate-ribose)) are covalently attached to proteins by PAR polymerase enzymes), recruitment of DNA repair factors and stabilization of chromatin for transcriptional regulation [15]. Importantly, since PARPi prevents repair of single strand breaks, causing DNA destabilization and eventual double strand breaks, cancer cells with deficient double strand repair (HRR) are particularly sensitive to PARPi [16]. Accordingly, based on preclinical and clinical results, in 2014 the US Food and Drug Administration (FDA) approved the first PARPi (i.e., olaparib) for treatment of patients with germline BRCA-mutated advanced ovarian cancer, who have been treated with three or more prior lines of chemotherapy. Since 2017, three PARP inhibitors (i.e., olaparib, rucaparib and niraparib), have received FDA approval in the ovarian cancer recurrent setting as maintenance therapy following platinum-based therapy [17–19].
Although several clinical trials are currently underway investigating the clinical efficacy and safety of PARPi for various human malignancies, limited preclinical and clinical information is currently available on the potential activity of olaparib in CC patients [20]. Accordingly, in this study, we evaluated the preclinical activity of olaparib against multiple homologous recombination competent (HRD) primary CC cell lines (i.e., both squamous and adenocarcinoma) and xenografts. Furthermore, we also investigated possible mechanisms behind CC sensitivity to PARPi and elucidated the correlation between sensitivity to olaparib and PARylation activity.
Study approval was obtained from the Institutional Review Board (IRB), and all patients signed consent prior to tissue collection according to the institutional guidelines. Nine primary CC cell lines (Table 1) were established from fresh tumor biopsy samples and maintained at 37 °C, 5% CO2 in Keratinocytes-SFM (Gibco®, Life Technologies™), supplemented with prequalified human recombinant Epidermal Growth Factor 1–53 (EGF 1–53), Bovine Pituitary Extract (BPE), 10%, 1% penicillin/streptomycin (Mediatech, Manassas, VA), and 1% Fungizone (Life Technologies, Carlsbad, CA). Briefly, cervical tumor biopsies were obtained from all patients and viable tumor tissue was mechanically minced under sterile conditions in enzyme solution [0.14% Collagenase Type I (Sigma St. Louis, MO) and 0.01% DNAse (Sigma, 2000 KU/mg)] in RPMI 1640, and incubated on a magnetic stirring apparatus 40’ at room temperature. The resultant cell suspension was washed in RPMI 1640 plus 10% FBS and then washed in PBS. Tumors were staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Patient characteristics are noted in Table 1
Table 1
Characteristics and demographic data of cervical cancer cell lines.
Cell line
Age
RACE
FIGO stage
Histology
HPV
CVX3
35
B
IB2
SCC
16
CVX4
40
W
IIA
SCC
16
CVX5
42
W
IB2
SCC
18
CVX7
22
H
IB2
SCC
16
CVX8
29
W
IB1
SCC
16
ADX1
33
W
IB
ADSQ
18
ADX2
33
B
IB
ACA
18
ADX3
25
W
IB
ACA
18
ADX4
47
B
IB
SCC
45
Homologous recombination deficiency (HRD) evaluation in CC cell lines
Log2-ratios of read counts in exonic intervals in whole exome sequenced (WES) tumor and normal samples [21], were tabulated (Figure 1S). Intervals were determined from high coverage regions in the normal samples, and intervals that did not overlap with RefSeq annotations were removed, to ensure remaining intervals corresponded to known genic loci. SNP allele frequencies were calculated in these exonic intervals, using SNPs defined in the phase 3 1000 Genomes dataset (Figure 2S). The log2-ratios and allele frequencies were used to assess HRD status for each sample using an ad hoc scoring algorithm, similar to the one used in the ARIEL2 trial [22].
Immunoblotting and antibodies
Cells were washed twice in ice-cold PBS and harvested with radioimmunoprecipitation assay buffer (RIPA) (50 mmol/L Tris–HCl, pH 8, 150 mmol/L NaCl, Triton X-100 1%, Na deoxycholate 0.5%, SDS 0.1%, MgCl 5 mmol/L in H2O) supplemented with Protease and Phosphatase Inhibitor (cat#78430, Thermo Fisher Scientific). Protein concentrations were measured by BCA Protein Assay Kit (Pierce™ #23225) to ensure equal loading. Proteins were denatured at 95°C for 5 minutes in Laemmli sample buffer (S3401; Sigma-Aldrich) and then resolved in SDS-PAGE electrophoresis, transferred on nitrocellulose, and blotted with corresponding antibodies. The antibodies used for western blotting were as follows: PAR (#4336, Trevigen), PARP (#9532, Cell Signaling Technology, Inc.), and GAPDH (#2118, Cell Signaling Technology, Inc.).
Cell viability assay
CC cell lines were plated at log phase of growth in 6-well tissue culture plates at a density of 80,000–100,000 cells/well. After 24 hours of incubation, cells were treated with Olaparib (AZD2281, LYNPARZA™, AstraZeneca) at a concentration of 0, 0.15, 1.5, 3, 12 μM. 72 hours after drug treatment, cells were harvested in their entirety, centrifuged and stained with propidium iodide (2 μl of 500 μg/ml stock solution in PBS). Count was performed using a flow-cytometry based assay to quantify percent viable cells as a mean ± SEM relative to untreated cells as 100% viable control. A minimum of three independent experiments per cell line was performed.
Cell-cycle analysis
After 48h incubation at the conditions described in Figure 4, cells were harvested and washed with ice-cold PBS, fixed in ice-cold 70% ethanol at −20°C for a minimum of 30 minutes to overnight. Subsequently, cells were washed in PBS, incubated with ribonuclease A (100 μg/ml) for 5 minutes at room temperature and stained with propidium iodide (20 μg/ml) in PBS. Cell-cycle phase distributions were analyzed with Flow-Jo software program (v. 8.7).
A) Cell cycle assay on CVX5 after 48h Olaparib treatment at the following concentrations: 0.15, 1.5, 3 μM (p=0.00005) B) Cell cycle assay on CVX8 (representative resistant cell line) after 48h Olaparib treatment at the following concentrations: 0.15, 1.5, 3 μM (p>0.05).
Annexin V-FITC/PI double staining
Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) double staining was used to quantify apoptosis. Adherent cells were incubated with 0, 0.15, 1.5, 3 μM of olaparib for 72 hours, then harvested and collected. Cells were washed twice with ice-cold PBS and resuspended in 1× Binding Buffer at a concentration of 1×106 cells/ml. 5 μl of Annexin V-FITC and 5 μl of propidium iodide were added to 100 μl of the cell suspension. After 15 minutes of incubation, 400 μl of Binding Buffer were added to each cell suspension. Cells were analyzed by flow cytometry within 1 hour.
siRNA transfection
Cells were plated in 6 well plate in Keratinocytes-SFM (Gibco®, Life Technologies™), supplemented with prequalified human recombinant Epidermal Growth Factor 1–53 (EGF 1–53), Bovine Pituitary Extract (BPE), 1% penicillin/streptomycin (Mediatech, Manassas, VA), and 1% Fungizone (Life Technologies, Carlsbad, CA). 70–80% confluent cells were subjected to transfection. PARP1 siRNA and negative control siRNA were purchased from Ambion®, Life Technologies™. Briefly, the siRNA was incubated with Lipofectamine™ RNAiMAX reagent (Invitrogen, CA, USA) in OptiMEM™ medium for 20 minutes, then added to a monolayer of cells in Keratinocytes-SFM without antibiotics. Twenty-four hours after the transfection, cells were treated with scalar amounts of olaparib ranging from 1.5 μM to 400 μM. Cells were then counted by flow cytometry.
Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)
RNA was obtained from cells after 48 hours of incubation with olaparib (Table 1S) using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA (5 μg) was reverse-transcribed using Superscript III (Invitrogen). Quantitative PCR was carried out to evaluate the expression level of PARP-1 (PARP-1, Assay ID: Hs00242302_m1, Applied Biosystems) in all samples with a 7500 Real-Time PCR System (Applied Biosystems) following the manufacturer’s protocol. Each reaction was run in duplicate. The internal control GAPDH (Assay ID: Hs99999905_ml, Applied Biosystems) was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle (Ct) method was used for the calculation of amplification fold as specified by the manufacturer. Analyses were performed using SDS software 2.2.2 (Applied Biosystems/Life Technologies).
In vivo treatment
The in vivo antitumor activity of olaparib was tested in xenograft models. Briefly, four to six-week-old CB17/SCID mice were given a single subcutaneous injection in the abdominal region of 7 × 106 CVX5 cells in approximately 300 μl of a 1:1 suspension of sterile PBS containing cells and Matrigel® (BD Biosciences). Xenografted mice were randomized into treatment groups (6 mice each group) when mean tumor burden was 0.15–0.25 cm3, and dosing (vehicle PO or olaparib 10 mg/kg BID, PO) was delivered to the CVX5 xenografts for 4 weeks (7 days/week). Drug dosage was chosen according to previous studies [23, 24]. Tumor and weight measurements of each mouse were recorded twice weekly. Mice were humanely euthanized when tumor volume reached 1.5 cm3 using the formula (width2 × height)/2. Animal care and euthanasia were carried out according to the rules and regulations as set forth by the Institutional Animal Care and Use Committee (IACUC).
Statistical analysis
Statistical analysis was performed using Graph Pad Prism version 8 (Graph Pad Software, Inc. San Diego, CA). The inhibition of proliferation in the CC cell lines after exposure to olaparib was evaluated by the two-tailed unpaired student t-test. Unpaired t-test was used to evaluate significant differences in the tumor volumes at specific time points in the in vivo experiments. Overall survival data was analyzed and plotted using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Differences in all comparisons were considered statistically significant at p-values < 0.05.
To evaluate the potential of PARP inhibitors on CC, we investigated the in vitro effects of olaparib on the growth of 9 primary CC cell lines using flow cytometric-based assay as described in the methods. As shown in Figure 1A, ,1B,1B, after 72 hours of incubation with increasing concentrations of olaparib, we found a progressive, dose-response decrease in cell proliferation in 33% of CC lines tested, with a significant difference in IC50 values between the sensitive and resistant group (p= 0.0012).
A)In vitro proliferation assay overview of the established primary CC cell lines (n=9) B) Violin scatter dot plot representing grouped sensitive cell lines and resistant cell lines (p=0.0012) C) Western blot analysis displaying basal expression of PARP, PAR, and GAPDH in all nine CC cell lines.
Sensitivity to olaparib is strongly correlated to PARP activity
To better understand the mechanisms behind the sensitivity to olaparib in a subset of primary CC, we analyzed PARP and PAR basal expression in all nine CC cell lines as well as their mutation spectrum (i.e., HRD), as defined in the methods section. None of the tested CC cell lines demonstrated HRD. Indeed, within the nine CC cell lines, genomic loss of heterozygosity (LOH) results ranged from 0–12.3% (Table 2S), which falls short of the initial ARIEL2 cutoff of 14% (and the current revised cutoff of 16%) used to classify a tumor as HRD [22]. In contrast, as demonstrated in Figure 1C, using immunoblot (i.e., cells lysates were loaded in order from the most sensitive to the most resistant CC based on IC50 values previously obtained by flow cytometric-based assay) we found a direct correlation between basal expression level of PARP activity (PAR) and sensitivity to olaparib treatment. Indeed, CVX5, CVX1 and CVX3 (i.e., the 3 CC primary cell lines with the higher PARP expression of both PARP isoforms 116 and 89 kDa), consistently demonstrated the higher sensitivity to olaparib exposure in the in vitro experiments.
Silencing of PARP-1 elicits resistance to olaparib
To evaluate further the correlation between PARP-1 activity and sensitivity of CC to olaparib we transiently transfected CVX5 cells with PARP-1 siRNA and negative siRNA control as described in materials and methods section. After 72 hours of olaparib treatment, IC50 values of either PARP-1 siRNA and negative control siRNA transfected CVX5 cells were evaluated through flow cytometric-based assay as described in Methods. Validation of PARP-1 mRNA silencing in tumor cells was confirmed with q-real time PCR (Table S1). As shown in Figure 2, CVX5 cells transfected with PARP-1 siRNA from sensitive become highly resistant (i.e., IC50 from 8.69 μM to 513.2 μM) to olaparib treatment (p=0.0063).
In vitro proliferation assay in PARP-1 silenced CVX5 cell line versus non-silenced control (p=0.0063).
Olaparib triggers apoptosis of CC in a dose-dependent manner
To gain better insight into the mechanism of PARPi activity, CVX5 was exposed to increasing concentration of olaparib (0.15, 1.5, 3 μM) for 48 hours before being harvested for Annexin V/PI staining. As shown in Figure 3, we demonstrated that olaparib at the dose of 1.5 μM and 3 μM induced apoptosis in 18% to 20% of cells, respectively, and tardive apoptosis in an additional 27.5% of cells (p<0.0001).
Up Left (UL) and Up Right (UR) quadrants show single positive events for FL1-H (ANNEXIN V-FITC) and double positive events for FL1-H and FL2-H, respectively. Double positive events stand for tardive apoptosis, corroborated by the absence of events in Down Right (DR) quadrant (single positive for FL2-H representing cell necrosis) (p<0.0001).
Olaparib sensitivity is associated with G2/M cell cycle arrest
We next examined the cell cycle profiles of CVX5 (i.e., a representative olaparib-sensitive CC cell lines) and CVX8 (a representative olaparib-resistant CC cell line) after 24 hours of olaparib treatment. As shown in Figure 4A, starting at 1.5 μM of olaparib, 67.7% of CVX5 cells demonstrated a G2/M cell cycle arrest (in comparison to non-treated cells (i.e., 22.3%) (p=0.000061). This percentage increased at the dose of 3 μM olaparib (78.3%) (p=0.00005). In contrast, as demonstrated in Figure 4B, CVX8 cell cycle was not affected by olaparib treatment at any dose tested (16.2% cells in G2-M non-treated cells vs 13.5% cells in G2-M after 3 μM olaparib treatment) (p>0.05).
Olaparib PARP inhibition and PARP trapping on sensitive CC
Next, we analyzed PARP-1 and PAR expression in CVX5 cells by immunoblotting assay after exposure to different doses of olaparib (0.15 μM – 1.5 μM) at two different time points (24–48 hours). As shown in Figure 5, PARP expression increased after exposure to 1.5 μM olaparib at both time points while no significant variation was detected in PARP-1 mRNA expression level at 24 or 48 hours (Table S1). A dramatic reduction in PAR levels was detected at both doses of olaparib (0.15 and 1.5 μM) (Figure 5).
Western blot analysis displaying expression of PARP, PAR, and GAPDH in CVX5 cells after 24–48 hours of treatment with 0.15 and 1.5 μM Olaparib.
Olaparib impairs CVX5 xenograft tumor growth in vivo
The in vivo effects of olaparib was determined by establishing xenografts from the primary CVX5 CC cell line. Briefly, after the tumors had reached the goal size, animals were randomized into treatment groups and treated as described in Materials and Methods. Tumor size was assessed weekly and mice were sacrificed if tumors became necrotic, reached a volume of 1.5 cm3, or mice appeared to be in poor health. Twice daily oral dose of olaparib 50 mg/kg was well tolerated with no clear impact on body weight compared with vehicle control (data not shown). As shown in Figure 6, mice undergoing olaparib treatment exhibited a significantly slower rate of tumor growth, compared to vehicle control starting at day 12 (p=0.0017). Furthermore, the overall survival was significantly prolonged in the treated group (Log Rank Mantel-Cox test p=0.008).
The inhibition of PARP was initially demonstrated to determine ‘synthetic lethality’ in cancer patients harboring specific DNA repair defects, (i.e., BRCA1 or BRCA2 (BRCA1/2) mutations) causing deficiency in the cell homologous recombination (HR) repair system [25, 26]. Accordingly, initial FDA approval was restricted to the treatment of patients harboring deleterious or suspected deleterious germline BRCA-mutated (gBRCAm) advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy. More recently, however, PARPi approval was expanded to maintenance therapy for patients with platinum-sensitive relapsed ovarian cancer, who responded to their second line regimen, regardless of BRCA1 or BRCA2 (BRCA1/2) mutation status [27] [28]. This broader use of PARPi stems from the evidence that tumors that share molecular features with BRCA-mutant tumors (i.e., BRCAness) also exhibit different levels of defective homologous recombination DNA repair, and therefore will respond to PARP inhibition [29]. Importantly however, recent results from large prospective randomized clinical trials have demonstrated significant PARPi clinical activity also against patients harboring HR-competent/BRCA wild-type tumors [30].
Unfortunately, while olaparib, rucaparib and niraparib are currently FDA-approved in ovarian cancer and multiple clinical trials are currently evaluating PARPi as single agents or in combination against multiple human tumors, limited information is currently available on the role of olaparib in CC patients. Accordingly, in this study, we thoroughly investigated the preclinically activity of olaparib against multiple primary CC cell lines in vitro and in vivo.
We found three of the nine primary CC cell lines to be highly sensitive to olaparib exposure with a cutoff IC50 value < 2μM [31]. To gain further insight into the molecular characteristics making these CC cell lines sensitive to olaparib treatment we evaluated their mutation spectrum (i.e., HRD), as well as their level of PARP1 expression [20, 32], and the potential role played by PARylation. Using the ARIEL2 study cutoff of 14% (and the current revised cutoff of 16%) used to classify a tumor as HRD [22], we found none of the tested CC cell lines to demonstrate HRD. Importantly, we found the level of PARylation but not PARP1 expression in the tumors to consistently correlate with CC cell line sensitivity to olaparib. To prove the correlation between PARylation overexpression and olaparib sensitivity was causative, we downregulated PARP1 mRNA through PARP1 siRNA transfection in a representative cell line (i.e., CVX5 cell line) and analyzed the IC50 values in comparison to transfected CVX5 with a universal negative control siRNA. We found CVX5 transfected with PARP1 siRNA to gain high resistance to olaparib treatment (p=0.0063), confirming that PARP activity (PAR) is of utmost importance in determining olaparib sensitivity in CC cell lines. These results are similar to the results obtained by Michels et al., who also found a positive correlation between cellular PARylation levels and sensitivity to PARP inhibitors in non-small cell lung carcinoma cell lines [33]. Moreover, in agreement with our results, other groups demonstrated that in the absence of functional HR, PARP1 or PARP2 knockout cells are resistant to PARP inhibitors [34, 35]. Taken together, these data combined with our findings in CC strongly suggest that determination of the level of PARP1 protein activity (i.e., PAR expression), may represent a biomarker potentially able to identify the most sensitive CC patients for treatment with PARPi. Accordingly, testing the possible link between PARP expression/activity and sensitivity to PARP inhibitors in the clinical setting may be warranted in future CC studies.
To better understand the functional mechanisms of olaparib in inhibiting CC cell growth, we performed cell cycle analysis experiments. We found olaparib, in a dose-dependent manner, to consistently arrest cell cycle in G2/M phase in all sensitive cell lines, ultimately preventing cells to going through the G1 phase. In contrast, no detectable alteration was found in the cell cycle of olaparib-resistant CC cell lines (i.e., CVX8). This effect of olaparib, as previously demonstrated in ovarian cancer, is explained by the PARP trapping mechanism, by which PARP inhibitors induce the formation of cytotoxic PARP–DNA complexes, preventing DNA replication [34]. When we investigated the mechanism of cell death in the CC cell lines exposed to olaparib, we found that only less than 1% of total cells demonstrated necrosis, corroborating the result that olaparib triggers and induces apoptosis in olaparib-sensitive CC cell lines.
To further elucidate the mechanism of action of olaparib against PARP, we analyzed PARP-1 and PAR protein expression in a representative cell line (i.e., CVX5) during olaparib treatment. Our immunoblot experiments clearly demonstrated a dose dependent increase of PARP1, as main consequence to olaparib exposure, further supporting an olaparib-induced PARP trapping phenomenon. In agreement with this interpretation, PARP-1 mRNA levels were not increased in any of the condition tested in any CC cell line. Taken together, our results support the notion of PARP-1 accumulation in cells treated with increasing concentrations of olaparib as main mechanism of action in CC. Importantly, when we evaluated the activity of olaparib in vivo in xenografted animals injected with CVX5, our result were confirmatory of the in vitro results with significant impairment of CVX5 tumor growth, and a significant increase in animal overall survival (p=0.008).
In conclusion, we demonstrated in vitro and in vivo activity of olaparib in a significant subset of CC primary cell lines and suggest that PAR expression may represent a novel biomarker for the potential prediction of PARPi response in patients with CC. Future studies with PARPi used alone or in combination with other targeted agents in patients with CC resistant to standard treatment modalities are warranted.
HIGHLIGHTS
A subset of primary CC cell lines is highly sensitive to olaparib in vitro and in vivo
High PARylation activity correlates with sensitivity to olaparib in CC cell lines
Silencing of PARP-1 reverses CC cell line sensitivity to olaparib and induce resistance
Preclinical studies demonstrate poly(ADP-ribose) polymerase (PARP) inhibition augments apoptotic response and sensitizes cervical cancer cells to the effects of cisplatin. Given the use of cisplatin and paclitaxel as first-line treatment for persistent or recurrent cervical cancer, we aimed to estimate the maximum tolerated dose (MTD) of the PARP inhibitor veliparib when added to chemotherapy.
Patients and methods
Women with persistent or recurrent cervical carcinoma not amenable to curative therapy were enrolled. Patients had to have received concurrent chemotherapy and radiation as well as possible consolidation chemotherapy; have adequate organ function. The trial utilized a standard 3 + 3 phase I dose escalation with patients receiving paclitaxel 175 mg/m2 on day 1, cisplatin 50 mg/m2 on day 2, and escalating doses of veliparib ranging from 50 to 400 mg orally two times daily on days 1–7. Cycles occurred every 21 days until progression. Dose-limiting toxicities (DLTs) were assessed at first cycle. Fanconi anemia complementation group D2 (FANCD2) foci was evaluated in tissue specimens as a biomarker of response.
Results
Thirty-four patients received treatment. DLTs (n =1) were a grade 4 dyspnea, a grade 3 neutropenia lasting ≥3 weeks, and febrile neutropenia. At 400 mg dose level (DL), one of the six patients had a DLT, so the MTD was not reached. Across DLs, the objective response rate (RR) for 29 patients with measurable disease was 34% [95% confidence interval (CI), 20%–53%]; at 400 mg DL, the RR was 60% (n =3/5; 95% CI, 23%–88%). Median progression-free survival was 6.2 months (95% CI, 2.9–10.1), and overall survival was 14.5 months (95% CI, 8.2–19.4). FANCD2 foci was negative or heterogeneous in 31% of patients and present in 69%. Objective RR were not associated with FANCD2 foci (P =0.53).
Conclusions
Combining veliparib with paclitaxel and cisplatin as first-line treatment for persistent or recurrent cervical cancer patients is safe and feasible.
Poly(ADP-ribose)polymerase inhibitors (PARPis) have shown promising activity in patients with BRCA1/2 mutation-associated (BRCA1/2MUT+) ovarian and breast cancers. Accumulating evidence suggests that PARPi may have a wider application in the treatment of sporadic high-grade serous ovarian cancer, and cancers defective in DNA repair pathways, such as prostate, endometrial, and pancreatic cancers. Several PARPis are currently in phase 1/2 clinical investigation, with registration trials now being designed. Olaparib, one of the most studied PARPis, has demonstrated activity in BRCA1/2MUT+ and BRCA-like sporadic ovarian and breast cancers, and looks promising in prostate and pancreatic cancers. Understanding more about the molecular abnormalities involved in BRCA-like tumors, exploring novel therapeutic trial strategies and drug combinations, and defining potential predictive biomarkers, is critical to rapidly advancing the field of PARPi therapy and improve clinical outcomes.Keywords: parp inhibitor, brca-like cancers, brca1/2 mutation, brca1/2 mutation-associated cancersGo to:
introduction
Progress has been made over the past two decades in the diagnosis, treatment, and prevention of cancer. A key component of progress in women’s cancers was the cloning of the BRCA1 and BRCA2 genes [1, 2] and reporting of The Cancer Genome Atlas’ (TCGA) comprehensive molecular analyses of high-grade serous ovarian cancer (HGSOC) and breast cancers [3, 4]. This knowledge is being translated into clinical opportunities through application of these new molecular definitions to tailor therapeutics uniquely to the individual patient.
Knowledge of BRCA1/2 mutation status in a patient has gone from a research question to demonstrated clinical utility directly affecting patient care. Dissection of their normal roles, both critical in normal DNA damage and repair, has led to better understanding of how their loss may cause or alter the course of cancer. Interestingly, neither knock-out nor knock-in models have demonstrated BRCA-1 or -2 to be independently causative in cancer development. They are embryonically lethal in knock-out settings, like many other tumor-suppressor genes [5]; selected knock-out is complementary to second genomic hits. The data for causality come from epidemiologic studies that define a tight relationship between deleterious BRCA-1 and -2 mutations (BRCA1/2MUT+) and development of breast and ovarian cancers [6], and increasingly with other cancers [7]. The seminal advance since the cloning and recognition of the relationship between loss-of-function mutations and breast and ovarian cancers is the identification, validation, and application of new biologically important molecular targets, poly-ADP ribose polymerase (PARP)-1 and PARP family members, and other proteins involved in homologous recombination (HR) repair of DNA damage.
DNA damage repair pathways
Six primary pathways of DNA repair have been identified [8]. They are variably used to address single- and double-stranded DNA break damage (SSB; DSB) from a variety of mechanisms of injury (Figure (Figure1);1); current results suggest pathway interaction and interdependence. Normal functions, such as cellular metabolism with associated generation of free oxygen radicals and reactive intermediates, ultraviolet light, therapeutic and ambient radiation, chemicals, and day-to-day replication errors, are common factors in the generation of DNA errors [9]. The function of the primary DNA repair pathways begins with sensing DNA damage, followed by recruitment of proteins involved in building the repair complexes [9]. Absence, reduction, or dysfunction of proteins in these pathways can be associated with loss of function of proper DNA repair. Four of the six repair pathways sense single-strand damage. HR, a high fidelity system, and nonhomologous end-joining (NHEJ), lower fidelity, are the two DSB repair programs [8]. BRCA1/2 mediate potentially rate-limiting events in HR [10]. It is now estimated that at least 15% of HGSOC occur in women with germline BRCA1/2MUT+, and another nearly 35% may have acquired defects in the HR pathway, including silencing by methylation, mutation in other repair genes, and activation of pathway inhibitors [3, 11].
Double-strand break repair and single-strand break repair with poly(ADP-ribose)polymerase inhibitors (PARPis).
Multiple studies suggest that the loss of p53 function cooperates with the loss of BRCA1/2 in tumorigenesis [12, 13]. The normal function of p53 is to recognize DNA damage and arrest cell cycle to either allow repair or to shut the cell down [14]. Incomplete or inadequate DNA repair thus triggers cell death in normal cells. TCGA [4] describes molecular similarities between HGSOC and triple-negative breast cancers (TNBCs), including dysregulation of the p53 and Rb checkpoints, leading to alterations in the expression of cell proliferation genes, DNA synthesis, DNA damage repair, cell cycle regulation, and apoptosis. p53 mutations are found in nearly 90% of HGSOC and in 80% of TNBC, both cancers with BRCA1/2 loss-of-function cohorts [3, 4, 15]. Chromosome breaks caused by loss of BRCA1/2 function activate p53-dependent checkpoint controls and/or apoptosis to prevent tumor formation. Selective pressure favors loss of p53 function to allow cell proliferation [16]. Mutant p53 facilitates G2/M transition, and cells acquire and propagate unrepaired DNA damage.
Loss of HR repair caused by loss of BRCA1/2 function leaves the cell needing alternative methods for DNA damage repair. SSB base excision repair (BER) is a primary back-up system for HR loss in response to BRCA1/2MUT+ [10]. The rate-limiting enzyme in BER, PARP-1, identifies the site of DNA injury and recruits repair complexes [17]. Recently, PARP-1 has been shown also to regulate NHEJ activity by holding this poor fidelity pathway in check [18], and to guide repair by forming PARP/DNA adducts [19]. These varied actions of PARP-1 form the increasingly strong basis for development of the PARP inhibitor class of agents (PARPi).Go to:
biology and beyond: parp inhibition
PARP-1 is a highly conserved enzyme focused to assist in the maintenance of genomic integrity [20]. It collaborates with PARG, polyADPribose glycohydrolase, required for hydrolysis and release of single-ADP-ribose moieties [20]. It has numerous other functions, including its cleavage and involvement in apoptosis, gene regulation through histone modification, and DNA decondensation for higher order chromatin function [21] and DNA repair [22]. The PARP-1 enzyme has been implicated in signaling DNA damage through its ability to recognize and rapidly bind to DNA SSB [23]; it also has been shown to participate in controlling the telomere length and chromosome stability [17, 24].
PARP-1 mediates BER by recruiting the scaffolding proteins XRCC1, DNA ligase III, and DNA polymerase ß [22]. The importance of PARP-1 in HR was shown in knock-out studies by a spontaneous increase in nuclear RAD51 focus formation [25], an event that signals active DSB repair. DNA-bound activated PARP-1 uses nicotinamide adenine dinucleotide (NAD+) to polyADPribosylate nuclear target proteins, the site of DNA damage, including topoisomerases, histones, and PARP-1 itself, to signal the need for both DNA SSB and DSB repair [26]. This observation suggests loss of PARP-1 activity where HR is compromised would lead to adverse consequences for the tumor cell.
New findings implicate PARP-1 as a negative regulator of NHEJ. Patel et al. [18] reported that PARP inhibition induces phosphorylation of DNA-dependent protein kinase cs (DNA-PKcs), a rate-limiting step in NHEJ activation. PARP-1-directed NHEJ may occur more selectively in HR-deficient cells where there is a default to secondary pathways. Implications of this include reversal of the genomic instability reported in HR-deficient cells after PARP inhibition. Murai et al. [19] showed PARP inhibitors trap PARP-1 and -2 at damaged DNA where the PARP–DNA complexes were more cytotoxic than unrepaired SSB, implicating PARPi as direct DNA poisons.
BRCA-like behavior and HR dysfunction
Understanding DNA repair biology has allowed us to identify patient subsets with high potential for response PARPi treatment. The marked susceptibility of patients with BRCA1/2MUT+-associated cancers has validated BRCA1/2MUT+ as a predictive biomarker for PARPi response [27]. Tumors in patients with germline BRCA1/2MUT+ contain a second, somatic loss of BRCA1/2, following the Knudson Hypothesis [28]; this occurs as a result of genomic injury and generally incorporates part or all of the second BRCA allele. This leaves the tumor tissue homozygous null for functional BRCA1/2, with impaired HR function. Fong et al. [27] were the first to confirm this link clinically, demonstrating that BRCA1/2MUT+-associated breast, ovarian, and prostate cancer patients receiving the olaparib had a 63% likelihood of clinical benefit. This led to the broad recognition of HR dysfunction (HRD) as a functional biomarker, and opened the door to examine phenocopy susceptibility. Phenocopy patients, those with HRD not caused by BRCA1/2MUT+, are those described as having BRCA-like behavior [29].
BRCA-like behavior has both molecular and clinical characteristics. Many mechanisms reducing BRCA1/2 function and resulting in BRCA-like behavior have been identified. Examples include BRCA1 promoter methylation [11–35% of epithelial ovarian cancers (EOCs)], Fanconi F (FANCF) methylation (5∼20%), and loss or reduction in FANCD2 [30], or other proteins necessary for HR [31, 32]. Nearly always associated with this level of HRD is an obligate mutation in p53 and frequent c-myc amplification. Loss of function of the suppressor gene, PTEN, has been shown to yield BRCA-like behavior, more common in breast and prostate cancers [33, 34]. Coexpression of BRCA1MUT+ and loss of PTEN protein expression were reported to occur in 82.4% of 34 breast tumor biopsies, suggesting that PTEN loss may be a common contributing event causing HRD [33]. Increased PARPi susceptibility was shown in a series of cell lines with PTEN mutation or haploinsufficiency, confirmed in xenograft experiments using the PARPi, olaparib. There is also clinical evidence that olaparib may have a therapeutic utility in PTEN-deficient endometrioid endometrial cancer [35]. These studies provide evidence that PTEN loss of function is a potential predictive biomarker of PARPi responsiveness.
Common clinical manifestations complement the molecular characteristics of BRCA-like behavior. The first BRCA-like behavior identified is susceptibility to platinum and other DNA damaging agents. This was initially inferred from studies demonstrating improved long-term survival of women with BRCA1/2MUT+-associated EOC receiving platinum-based combination chemotherapy [36]. Intra- and inter-strand platinum-DNA crosslinks can create torsion on the double helix and lead to DSBs [31], requiring HR for proper and successful correction. Without repair, further genomic injury is sustained, leading to cell death. Reports also describe increased overall survival and progression-free survival (PFS) for mutation carriers receiving other DNA-damaging agents, such as pegylated liposomal doxorubicin (PLD) [37, 38]. Overall survival with PLD alone was nearly double that expected from large trials in a non-selected (general) population (median PFS 7.1 months; 95% CI 3.7–10.7), and similar findings were reported in a retrospective analysis of outcome following PLD in women who were BRCA1/2 germline mutation carriers and those considered not to harbor a germline mutation [38]. Subsequently, these characterizations have led to population evaluations, now suggesting that HRD occurs in up to 50% of HGSOC [11, 39, 40] and 20% of TNBC [41]. Dissection of these clinical and molecular data will inform further study design and improve therapeutic application of PARPi.Go to:
updating clinical applications of PARP inhibitors
Multiple PARPis are in clinical development as single agents and/or in combination therapy (Table (Table1).1). The most common PARPi chemistry is that of reversible NAD mimetics, with differences in bioavailability and molar equivalence of PARP enzyme inhibition. There are at least six agents under study in this class; iniparib (BSI-201) is another compound that is not a true PARPi [42]. The loss of BER capacity produced by PARPi has prompted evaluation of these drugs as potential enhancers of DNA damaging cytotoxic agents, such as alkylating agents or radiation therapy, leading to new directions for combination therapies [18, 19].
Table1.
Active PARP is under development
PARPi
Treatment
Cancer types
Phase
Olaparib (AstraZeneca)
-Monotherapy -Combinations with cytotoxic chemotherapy -Combinations with targeted agents -Combinations with RT
BRCA1/2MUT+ associated BrCa/OvCa, BRCA-like tumors, Advanced hematologic malignancies and solid tumors, Maintenance study following remission in platinum sensitive OvCa (pending)
I/II/III
Veliparib (Abbott)
-Monotherapy -Combinations with cytotoxic chemotherapy -Combinations with targeted agents -Combinations with RT
-Monotherapy -Combinations with cytotoxic chemotherapy
Advanced solid tumors
I
Niraparib (MK-4827) (TesaroBio)
-Monotherapy -Combinations (temazolomide)
Advanced hematologic malignancies and solid tumors, BRCA1/2MUT+ associated and HER2 negative BrCa, Maintenance study following remission in platinum sensitive OvCa (pending)
*OvCa, ovarian cancer; BrCa, breast cancer; RT, radiation therapy.
Initial dose-finding trials have demonstrated significant clinical activity of PARPi especially in BRCA1/2MUT+ breast and ovarian cancers [43–46]. This suggests that BRCA 1/2MUT+ is a genetic marker for targeted therapy, similar to other therapies targeted against loss-of-suppressor function mutations that have been shown to have clinical benefit. Angiogenesis inhibition provided benefit in germline Von Hippel Landau mutation-related renal clear cell cancer, shown to have a VHL-mediated hypoxia-inducing factor 1α-VEGF drive [47]. Similarly, activating mutations of RET are associated with the pathogenesis and vandetanib-sensitivity of medullary thyroid cancer [48]. Current clinical development for PARPi builds upon these observations. The patient populations targeted in PARPi clinical trials include patients with BRCA1/2MUT+ cancers, BRCA-like cancers, and those with recognized susceptibility to DNA-damaging agents, but without BRCA-like association, such as lung or pancreas cancers (Table (Table22).
Table 2.
Ongoing clinical trials of PARPis for other malignancies, except breast and ovarian cancers
Cancer type
Subtypes
PARP inhibitor
Phase
GI malignancies
Colorectal cancer
Veliparib + TMZ Olaparib + irinotecan
I/II
Pretreated colorectal cancer stratified by Microsatellite Instability (MSI)
Olaparib monotherapy
I/II
Gastric cancer
Veliparib + FOLFIRI
I/II
Gastric cancer with low ATM protein level
Paclitaxel +/− olaparib
II
Esophageal cancer
Olaparib + RT
I
Metastatic pancreatic cancer
Olaparib + Gemcitabine Veliparib + modified FOLFOX6 + gemcitabine + gemcitabine/IMRT Veliparib for BRCA or PALB2 mutated pancreatic cancer Gem/cis +/− veliparib
Initial phase I/II clinical trials demonstrated single-agent activity of olaparib in BRCA1/2MUT+ breast, ovarian, and prostate cancers, and recurrent HGSOC; [27, 44, 49], no single agent response data have yet been reported for CEP-9722 (Table (Table3).3). The study by Gelmon et al. [48] clearly showed that patients with platinum-sensitive HGSOC responded to olaparib without a BRCA1/2 germline mutation. Ledermann et al. [50] recently reported maintenance olaparib significantly improved PFS in a randomized, placebo-controlled, phase II trial in platinum-sensitive HGSOC following a response to two or more lines of platinum-based therapy [50]. They demonstrated a nearly doubling of median PFS post chemotherapy (8.4 versus 4.8 months) and a 65% reduction in risk of disease progression. An interim survival analysis [51] with 58% maturity showed difference between olaparib and placebo, notably in the BRCA1/2MUT+ with a hazard ratio (HR) of 0.18 (95% CI 0.11–0.31) and with a median PFS of 11.2 versus 4.3 months, respectively. Overall survival did not show difference in this group, (HR = 0.74; median: 34.9 versus 31.9 months) probably due to 22.6% of patients on placebo switched to olaparib. As a result of these findings registration trials are being developed with olaparib and other PARPi as maintenance therapy following treatment of platinum-sensitive relapsed ovarian cancer. These types of maintenance study may even be taken into front-line therapy for selected patients.
Table 3.
Single-agent activity with PARPi in phase I/II studiesa
The greatest clinical experience to date is with olaparib monotherapy. It generally well tolerated at doses of 400 mg twice daily in capsule formulation with many patients able to take the drug for several years. A new tablet formulation [56 ], reducing the number of pills that need to be taken is being assessed. PK data including AUC0−T and Cmin from 300 mg and 400 mg tablet doses matched or exceeded the 400 mg capsule dose, and 300 mg tablet is expected to be incorporated into further studies in mid-2013. PARPis have been tested in combination with various DNA damaging agents. Studies have shown clinical benefit and interactive adverse events, including bone marrow toxicity and fatigue [27, 43, 57]. Class-based adverse events also include fatigue, headache, nausea, and reflux in 25–40% of patients. Early reports also suggest a possible increased clinical benefit in combination therapy, that may out balance the toxicities [57, 58]. Continued follow-up and diligence are needed to define the risk of long term PARPi therapy.
Current therapeutic directions for PARPi are focused at designing combinations, determining optimal timing of therapy and breadth of application of this key class of agents to and beyond mutation carriers. Agents selected for the combination study include those likely to cause replication fork injury or further DNA damage, and anti-angiogeneic agents. Hypoxia was shown to cause DNA damage when a second DNA hit was included in a mouse model [59]. We exposed microvascular endothelial cells in vitro to the VEGF receptor antagonist, cediranib (AZD2171), in combination with olaparib, demonstrating a cooperative inhibition of angiogenesis (Kim and Kohn, unpublished data). Surprisingly, interactive anti-invasive activity was observed with this combination against a p53-mutant HGSOC cell line, OVCAR8. A phase I study of olaparib and cediranib showed clinical promise [60], and a multi-institutional randomized phase II study is in progress (NCT01116648). Additionally, a phase I study of continuous daily olaparib with bevacizumab was generally well tolerated in patients with advanced solid tumors [61].
Phase I/II studies are ongoing with PARPi and a variety of agents (Table (Table1).1). A phase I study of olaparib with carboplatin (AUC4/5) showed clinical benefit in 85% of 27 women with BRCA1/2MUT+-associated recurrent breast and ovarian cancers [58]. A randomized, phase II study of olaparib with paclitaxel (Taxol) and carboplatin (AUC4) followed by olaparib maintenance resulted in a significant improvement in PFS compared with paclitaxel, Bristol-Myers Squibb (New York) and carboplatin, Bristol-Myers Squibb (New York) (AUC6) alone in women with platinum-sensitive recurrent HGSOC (HR = 0.51; median PFS 12.2 versus 9.6 months) [62]. This suggests that combining olaparib with carboplatin required a dose modification of both drugs, illustrated the potential for toxicity interaction with DNA active agents. There was no difference in PFS during the period of chemotherapy in this trial; differences emerged in the maintenance phase. The optimal dosage, scheduling, and sequencing of PARPis and cytotoxic agents require carefully designed clinical trials linked to preclinical studies that specifically address the above issues.
This promising therapeutic potential has elicited considerable interest in clinical development of the PARPi class. Early clinical data also suggest that a BRCA-like gene expression profile may correlate with clinical responses to the platinum drugs in patients with sporadic EOC [63, 64]. Prospective validation and optimization of these signatures in a broad array of cancers, and appropriate selection of a patient population are imperative to achieve the full potential of PARPis.
challenges to PARP inhibitor development
The incorporation of targeted agents into therapy of BRCA1/2MUT+ and BRCA-like cancers presents challenges. First is development of a mechanism with which to identify patients who are most likely to benefit. Discovery and validation of predictive biomarkers is an active area of ongoing research. Biomarkers for patient selection or stratification are recommended by the US Food and Drug Administration for approval of new targeted drugs. Loss of BRCA1/2 expression, generally by demonstration of a deleterious germline mutation, is a validated predictive biomarker. Routine testing of patients is being increasingly adopted as up to 17% of patients with HGSOC, the most common form of ovarian cancer, have germline mutations [65]. However, BRCA1/2 mutation testing does not identify the full range of potentially susceptible patients, and it requires a validated predictive BRCA1/2 mutational testing tool. BRCA1/2 loss in the tumor by mutation or methylation may also be inferable by loss of BRCA1/2 protein expression demonstrated by immunohistochemical staining, leaving reduction in BRCA1/2 protein expression as a potential predictive tool [39].
The histone protein H2AX becomes rapidly phosphorylated and concatemerizes at nascent DNA DSBs [66]. This creates a focus for accumulation of DNA repair and chromatin remodeling proteins. DSBs can be labeled with an antibody to the phosphorylated form, γH2AX, and extent of DSB estimated from the number of labeled foci (Figure (Figure2)2) [66]. RAD51 is instrumental in initiation of assembly of HR repair proteins at the site of DNA injury [67]. Formation of nuclear RAD51 foci can be assessed by immunofluorescence and is a marker of HR competence. Formation of γH2AX and/or RAD51 foci after DNA damage has been suggested as pharmacodynamic biomarkers of PARPi activity; demonstrating that a change in these parameters early in treatment may be examined as potential predictive biomarkers. A phase 1 study of veliparib and topotecan showed an increase in γH2AX focus formation by immunofluorescence in circulating tumor cells from seven of nine patients [68], with no correlation to clinical outcomes. Inhibition of RAD51 focus formation by PARPi was shown in vitro in EOC ascites primary cultures and correlated with response to PARPi [69]. This suggests that the lack of RAD51 foci may indicate potential drug response [70].Open in a separate windowFigure 2.
γH2AX binds to DNA DSBs and RAD51 initiates repair protein assembly in the homologous recombination (HR) pathway.
Predictive biomarkers applied to readily available bioresources, such as archival tissue or non-tumor tissue, have been proposed. Changes in PAR (poly ADP Ribose) incorporation into peripheral blood mononuclear cell DNA were evaluated as a putative early on-treatment pharmacodynamic measure; while present, there was no relationship to clinical outcomes [57]. Basal levels of PAR vary in different cells, reflecting their relative capacity for DNA repair, and requiring demonstration of change in PAR concentrations over time. Hence, identifying an accurate measure of HR potential for application as a predictive biomarker remains necessary to guide administration of PARPi.
Dissecting and defining mechanisms of development of resistance to PARPis, and whether this portends potential collateral resistance to other DNA damaging agents is the second challenge. Acquisition of a secondary mutation in BRCA1/2 that allows BRCA1/2 gene read-through and yields a functional protein has been demonstrated in cell lines and some patients; this was correlated with loss of susceptibility to PARPi treatment [71]. A second, preclinically defined method of resistance is loss of function of 53bp1 [72], a key protein in the NHEJ pathway. Whether or not 53bp1 expression can be used as a selective or predictive biomarker is yet to be determined. Understanding the mechanism(s) of resistance to PARPi will lead to optimal application and sequencing of PARPi and platinum compounds. Studies are needed to evaluate outcomes to subsequent chemotherapies in patients who have received PARPis [73].Go to:
conclusion
Several PARPis are under investigation and it is anticipated that this novel and exciting new class of compounds will ultimately receive regulatory approval in select subsets of cancers. This class of agents has tolerable toxicity profiles and has been given to patients for long periods. Clinical benefit has been observed in patients with BRCA1/2MUT+-associated cancers and BRCA-like phenotypes in germline mutation-negative patients. It is for these patients, in particular, that predictive markers for HR deficiency and response to PARPi are needed, so that patients can be selected for therapy. Understanding more about the molecular abnormalities involved in BRCA-like tumors will be critical to advance the field of PARP inhibition therapy and in improving patient selection and consequent clinical outcomes.
Topoisomerase 1 inhibitors cure human cancer xenografts in animal models, more so than most other chemotherapy agents.
However, their activity in patients with cancer is modest.
Ongoing research is studying the optimal analogs that could reproduce animal data in the cancer population.
This article analyzes the clinical research with topoisomerase 1 inhibitors in ovarian cancer.
The first type I topoisomerase (Top1) inhibitors were found in the wood bark of Camptotheca acuminata, an oriental tree, the powder or injectable extracts of which have been used in traditional Chinese medicine.1 The class was named camptothecin (CPT) for the basic CPT compound (Fig. 1). Clinical studies of this group of drugs were initiated in the 1970s, and in the 1980s the Top1 enzyme was identified as the cellular target of CPT.2,3 Topoisomerases relax the DNA supercoiling and perform catalytic functions during replication and transcription.4 There are two classes of topoisomerases. Type I enzymes cleave one strand of DNA and type II cleave both strands. Six topoisomerase genes have been identified in mammalian somatic cells within these two classes. Type IA enzymes consist of Top3 a and Top3 b; type IB consist of Top1 and Top1mt (mitochondrial); and type IIA consist of Top2 a and Top2 b. CPT is an inhibitor of Top1. Top1 cleaves the DNA phosphodiester backbone, nicking one strand of the DNA duplex and forming a Top1-DNA reversible cleavage complex by covalent bonding of a tyrosine residue. Single-strand breaks induced by Top1 help untangle excessive DNA supercoils during DNA replication and transcription (Fig. 2).5,6 Top1 is essential for survival.
Catalytic topoisomerase II inhibitors in cancer therapy
The nuclear enzyme DNA topoisomerase II is a major target for antineoplastic agents. All topoisomerase II-directed agents are able to interfere with at least one step of the catalytic cycle. Agents able to stabilize the covalent DNA topoisomerase II complex (also known as the cleavable complex) are traditionally called topoisomerase II poisons, while agents acting on any of the other steps in the catalytic cycle are called catalytic inhibitors. Thus, catalytic topoisomerase II inhibitors are a heterogeneous group of compounds that might interfere with the binding between DNA and topoisomerase II (aclarubicin and suramin), stabilize noncovalent DNA topoisomerase II complexes (merbarone, ICRF-187, and structurally related bisdioxopiperazine derivatives), or inhibit ATP binding (novobiocin). Some, such as fostriecin, may also have alternative biological targets. Whereas topoisomerase II poisons are used solely for their antitumor activities, catalytic inhibitors are utilized for a variety of reasons, including their activity as antineoplastic agents (aclarubicin and MST-16), cardioprotectors (ICRF-187), or modulators in order to increase the efficacy of other agents (suramin and novobiocin). In this review, the mechanism and biological activity of different catalytic inhibitors is described, with emphasis on therapeutically used compounds. We will then discuss future development and applications of this interesting class of compounds.
Fig. 1. The catalytic cycle of DNA topoisomerase II. The ATPase domains of topoisomerase II are shown in light blue, the core domain in dark blue, and the active site tyrosine residue in red. The C-terminal domain of the enzyme is not included in the diagram since its orientation, with respect to the rest of the molecule, is not known. The catalytic cycle is initiated by enzyme binding to two double-stranded DNA segments called the G segment (in red) and the T segment (in green) (Step 1). Next, two ATP molecules are bound, which is associated with dimerization of the ATPase domains (Step 2). The G segment is cleaved (Step 3) and the T segment is transported through the break in the G segment, which is accompanied by the hydrolysis of one ATP molecule (Step 4). The G segment is then religated and the remaining ATP molecule is hydrolyzed (Step 5). Upon dissociation of the two ADP molecules, the T segment is transported through the opening in the C-terminal part of the enzyme (Step 6) followed by closing of this gate. Finally, the N-terminal ATPase domains reopen, allowing the enzyme to dissociate from DNA (Step 7). Data from Berger et al. (1996), Baird et al. (1999), Brino et al. (2000), and Hu et al. (2002). https://www.researchgate.net/profile/Alexandre_Escargueil3/publication/10638629/figure/fig1/AS:281505667534857@1444127589543/Fig-1-The-catalytic-cycle-of-DNA-topoisomerase-II-The-ATPase-domains-of-topoisomerase.png
Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that are involved in crucial aspects of cancer biology, including angiogenesis, cell survival, glucose metabolism and invasion. Intratumoral hypoxia and genetic alterations can lead to HIF-1α overexpression, which has been associated with increased patient mortality in several cancer types. In preclinical studies, inhibition of HIF-1 activity has marked effects on tumour growth. Efforts are underway to identify inhibitors of HIF-1 and to test their efficacy as anticancer therapeutics.
Targeting DNA topoisomerase II in cancer chemotherapy
Recent molecular studies have greatly expanded the biological contexts where Top2 plays critical roles, including DNA replication, transcription and chromosome segregation. Although the biological functions of Top2 are important for insuring genomic integrity, the ability to interfere with Top2 and generate enzyme mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed understanding the biological functions of Top2 are also being applied to understanding the details of drug action. These studies promise a more refined ability to target Top2 as an effective anti-cancer strategy.
An important reason why Top2 has held the interest of researchers studying cancer was the discovery that active anti-cancer drugs, notably etoposide and doxorubicin target Top21. These studies showed that most clinically active drugs that target Top2 generate enzyme mediated DNA damage2–4. Since etoposide and doxorubicin are highly active anti-cancer agents in many different settings, an identification of a critical target of these drugs was a major landmark in the pharmacology of anti-cancer drugs.
Recent work has shown that there may be contexts where the level of Top2 protein predicts clinical activity (as well as many contexts where it does not). With the understanding of mechanisms of drug action and improved patient survival rates has come the appreciation that clinical treatment with drugs targeting Top2 can lead to the dire consequence of secondary malignancies. An important goal of present and future work is to maximize therapeutic efficacy of therapy using Top2 targeting agents while minimizing the risks of secondary malignancy and other toxicities. This review highlights recent work that is relevant to maximizing the potential of Top2 as an anti-cancer drug target.
Inhibition of Top2 activity by anti-cancer agents
Drugs targeting Top2 are divided into two broad classes. The first class, which includes most of the clinically active agents including etoposide, doxorubicin, and mitoxantrone, lead to increases in the levels of Top2:DNA covalent complexes. Because these agents generate a “lesion” that includes DNA strand breaks and protein covalently bound to DNA, these agents have been termed Top2 poisons. A second class of compounds inhibits Top2 catalytic activity, but do not generate increases in the levels of Top2 covalent complexes. This second class of agents is thought to kill cells through elimination of the essential enzymatic activity of Top2 and is therefore termed catalytic inhibitors (Fig. 1).
Top2 can be inhibited at several different points in the enzyme reaction cycle, which can generate different biochemical and cellular consequences. One simple mode of inhibition is to inhibit a step early in the enzyme reaction cycle. For example, competitive inhibitors of ATP binding prevent strand passage, and do not generate enzyme mediated DNA damage. While agents such as novobiocin and coumermycin (not shown on the figure) inhibit both prokaryotic and eukaryotic Top2s, they are either less potent as well as relatively nonspecific (e.g., novobiocin) or are poorly taken up by mammalian cells (e.g., coumermycin). Similar effects would occur with inhibitors that prevent the binding of Top2 to DNA such as aclarubicin. Agents that prevent DNA cleavage by Top2, such as merbarone would also be expected to act as simple catalytic inhibitors. While merbarone clearly prevents DNA cleavage by Top2126, merbarone clearly affects other targets besides Top2. A second mode of inhibition is blocking the catalytic cycle after DNA is cleaved but prior to DNA religation. This mode of inhibition occurs for most currently used Top2 targeting agents including anthracyclines and epipodophyllotoxins, as well as for agents that target prokaryotic type II topoisomerases. These agents prevent enzyme turnover, and therefore greatly inhibit the enzyme catalytic activity, however, the clearest effect is the generation of high levels of Top2:DNA covalent complexes. Therefore, these inhibitors generate DNA damage, and interfere with many DNA metabolic events such as transcription and replication. Since agents of this class convert Top2 into an agent that induces cellular damage, they have been termed topoisomerase poisons. Top2 can be inhibited after strand passage is completed, but prior to ATP hydrolysis and dissociation of N-terminal dimerization. Bisdioxopiperazines such as dexrazoxane (ICRF-187) inhibit both ATP hydrolysis and maintain Top2 as a closed clamp 74. As is the case with Top2 poisons, bisdioxopiperazines inhibit Top2 catalytic activity mainly by blocking enzyme turnover. Although these agents are frequently termed catalytic inhibitors, they leave Top2 trapped on DNA, and may interfere with DNA metabolism in ways distinct from the inhibitors described in pathway (A). Nonetheless, since bisdioxopiperazines are relatively specific for Top2, they are the most commonly used catalytic inhibitors of Top2 in mammalian cells 143.
There are several lines of evidence indicating the importance of the distinction between Top2 poisons and Top2 catalytic inhibitors. Studies in yeast and mammalian cells demonstrated that resistance to Top2 poisons is recessive, i.e., presence of a drug resistant Top2 in the presence of a drug sensitive allele results in cells that are drug sensitive (reviewed in 5,6). The importance of enzyme mediated DNA damage is also demonstrated by observations that Top2 poisons rapidly elicit DNA damage responses such as ATM phosphorylation and activation of downstream damage responses7–9. Resistance to Top2 targeting drugs in mammalian cells is frequently associated with reduced expression of Top2 isoforms6, suggesting that resistance is mediated through a reduction in enzyme mediated DNA damage, rather than through enhancing available enzyme activity (where resistance would arise from increased expression of Top2 isoforms).
The generation of high levels of Top2 DNA covalent complexes has profound effects on cell physiology. Top2 poisons effectively block transcription and replication. DNA strand breaks are rapidly detected following treatment with Top2 poisons, and most of the strand breaks are protein linked, as expected10,11. Cells subsequently commit to apoptosis, in fact etoposide is a very commonly used agent to study apoptotic processes12.
The pattern of responses observed with catalytic inhibitors of Top2 differ from that observed with Top2 poisons, albeit with several important complications. Most catalytic inhibitors of Top2 are not specific for Top2 inhibition (see Box 1) with the exception of bisdioxopiperazines. While bisdioxopiperazines generate DNA damage responses following long exposure13, they do not produce a DNA damage response following short term exposure14–17. Importantly, in cell culture experiments, catalytic inhibitors of Top2 antagonize the toxicity of Top2 poisons18, indicating that the agents act by separable mechanisms. An important and still unanswered question is whether Top2 inhibitors that are not poisons might be active anti-cancer agents. This issue is addressed in the concluding sections of this review.
Box 1. Many different classes of compounds target Topoisomerase II
Drugs targeting topoisomerase II fall into two categories, Top2 poisons and Top2 catalytic inhibitors. Many Top2 poisons have demonstrated anti-cancer activity. Top2 poisons can be further sub-divided into intercalating and non-intercalating poisons. The intercalators are chemically diverse, and include doxorubicin and other anthracyclines, mitoxantrone, mAMSA, and a variety of other compounds that are not currently in clinical use such as amonafide and ellipticine5. Other than their ability to intercalate in DNA, there is no obvious chemical similarity that could explain the ability of these compounds to trap Top2. Importantly, some compounds, such as oAMSA and ethidium bromide have little ability to poison Top2, suggesting that intercalation of a small molecule is insufficient to trap Top2 as a covalent complex on DNA1,110. Some of the intercalating Top2 targeting drugs, notably the anthracyclines, produce a variety of effects on cells, including many effects that are independent of their action against Top2. For example, doxorubicin is known to produce free radicals, to cause membrane damage, and to induce protein:DNA crosslinks. Whether Top2 is the most important target of anthracyclines remains a controversial issue, (reviewed in 111), although some of the results presented in the text support the hypothesis that Top2 is the most relevant target for both clinical response and cardiotoxicity. For alternate hypotheses, see 112–114.
Several classes of compounds have been described that inhibit Top2 activity but do not increase DNA cleavage. Most prominent are the bisdioxopiperazines, which inhibit the enzyme ATPase activity non-competitively and trap Top2 as a closed clamp74,117,118. ICRF-187, a bisdioxopiperazine, is used as a cardioprotectant in some patients treated with anthracyclines. Other Top2 catalytic inhibitors include novobiocin119–121, merbarone122, and the anthracycline aclarubicin123. All three compounds have significant targets besides Top2121,124,125; therefore these compounds have not been useful in assessing the feasibility of using catalytic inhibitors of Top2 as an anti-cancer therapy. Merbarone has attracted interest because it is the only agent that has been found to inhibit Top2 cleavage of DNA but not affect protein:DNA binding126. QAP1 is a newly described purine analog that was rationally designed to target the Top2 ATPase activity127. This compound may be particularly useful in assessing the effects of catalytic inhibition of Top2. Several other catalytic inhibitors have been described, however, their detailed mechanism of action has not been explored.
The future of Top2 as a drug target
Is there a need for new and different Top2 drugs? The first answer to this question is a resounding yes, since Top2 targeting is clearly successful in a wide variety of contexts. It is clear from broiad clinical experience that Top2 targeting drugs can be safely and effectively combined with many other agents. The Top2 targeting drugs in clinical use were identified not based on their activity against Top2, but mainly on empirical anti-tumor activity. Therefore, it would be expected that rational screening would lead to potent and specific Top2 poisons. It would be very desirable to know if greater potency and specificity would enhance clinical response.
At the time etoposide and doxorubicin were approved for use, we did not know of the existence of Top2β. The results reviewed in this article suggest that the targeting of Top2β leads to several undesirable consequences and little clear benefit. The negative effects of targeting Top2β include the induction of cardiotoxicity, and potentially a major role in secondary malignancies. On the other hand, there are potential benefits of targeting Top2β, especially the ability to kill non-proliferating cells. While targeting Top2β may contribute to toxicity, it may also be important for eliminating cancer cells that function as cancer stem cells.
An important question is whether isotype specific Top2 poisons can be identified, since the two enzymes share catalytic mechanisms, and a great deal of amino acid homology in their catalytic domains. It has been previously suggested that the intercalators mAMSA and mitoxantrone confer cytotoxicity mainly due to targeting Top2β106. More recently, a novel intercalator NK314 has been reported to be highly specific for Top2α107,108. Toyoda and colleagues also suggested that etoposide and doxorubicin generate greater cytotoxicity by targeting Top2α. Taken together, these results suggest that agents specific for Top2α may possible, and may be useful for having both greater anti-tumor activity, and reduced toxicitiy.
The search for improved Top2 targeting drugs will require further advances in both the biochemistry and structural biology of drug action. While the structures that have already been determined have provided important insights into the biochemistry of Top2, the only structure of Top2 bound to a drug that has been determined is the ATPase domain of Top2 bound to ICRF-187109. The grail for understanding the biochemistry of a drug like etoposide is the determination of a ternary complex between drug, protein, and DNA. Hopefully, the structures of the breakage/reunion domains of Top2α and Top2β, especially their DNA bound forms, will be solved soon.
An interesting question related to drug development is whether catalytic inhibitors of Top2 might be active anti-cancer agents. Much of the literature on the action of Top2 poisons implicitly assumes that they inhibit Top2 activity. Compared to many other enzyme inhibitors, any of the currently described Top2 targeting agents has relatively poor potency (for example, the Ki of etoposide for Top2 is in the 5-20 μM range, the Ki for ICRF-193 is in the 1-2 μM range). The availability of crystal structures provides the tools for addressing whether Top2 inhibition will be a valuable strategy (and will provide tools needed to answer many important biological questions).
The recent biological insights in transcription, replication and checkpoint control also offer ways to better understand drug action and resistance. Since cancer cells can clearly present with altered topoisomerase levels, whether by amplification or changes in gene regulation, these alterations provide an opportunity for enhanced therapeutic index. Finally, active anti-cancer therapy requires an understanding of how cancer cells ‘make a living’, and topoisomerases clearly are central to many of these core biological functions.
At a glance
Top2 is the target of several important classes of anti-cancer drugs, including the epipodophyllotoxin etoposide, and the anthracycline doxorubicin.
Most clinically active drugs that target Top2 kill cells by trapping an enzyme intermediate termed the covalent complex. Therefore, the principal action of Top2 targeting drugs currently used are to generate enzyme mediated DNA damage.
A recent structure of the breakage reunion domain of Top2 bound to DNA has been determined. This structure is likely to be of great use in understanding the protein determinants of the action of drugs targeting Top2. A drug:protein:DNA ternary complex would be extremely valuable, but has not yet been determined.
Top2 mediated DNA damage is repaired by multiple pathways. The DNA damage includes DNA strand breaks and proteins covalently bound to DNA. Repair of Top2 damage requires double strand break repair pathways, and other pathways specific for the removal of protein:DNA adducts.
Sensitivity to Top2 targeting drugs depends in part on levels of Top2 protein. Cells overexpressing Top2 are hypersensitive to Top2 poisons while cells expressing low levels of Top2 are relatively drug resistant. Top2α is frequently co-amplified with ERBB2. This can lead to some tumors with elevated levels of Top2α.
An important side effect of targeting Top2 with Top2 poisons are secondary malignancies arising from drug induced translocations. Top2β may be the Top2 isoform that is most responsible for secondary malignancies caused by Top2 targeting drugs.
Anthracycline use is limited by cardiotoxicity. Although the mechanism of the cardiotoxicity is poorly understood, recent results suggest that anthracyclines acting against Top2β may contribute significantly to cardiotoxicity. There may be considerable benefit to developing Top2 targeting drugs specific for the Top2α isoform.
Catalytic inhibition of Top2 may also be a useful anti-cancer strategy. New compounds are being developed to test this possibility.
Goulet ML, Olagnier D, Xu Z, Paz S, Belgnaoui SM, Lafferty EI, Janelle V, Arguello M, Paquet M, Ghneim K, Richards S, Smith A, Wilkinson P, Cameron M, Kalinke U, Qureshi S, Lamarre A, Haddad EK, Sekaly RP, Peri S, Balachandran S, Lin R, Hiscott J.
A new modified form of Doxorubicin with different mechanism of action may provide antitumor activity while separating away the cardiotoxic effects
A colorful chemotherapy agent could be made less toxic. Jocelyn Kaiser. Science 03 Jul 2020: Vol. 369, Issue 6499, pp. 18 DOI: 10.1126/science.369.6499.18
……. But because anthracyclines can cause heart damage, physicians often avoid giving them to elderly patients. Many childhood cancers are treated with high doses of the drugs, but cardiac problems sometimes haunt survivors later in life, along with a risk of new tumors, which doctors have attributed to DNA damage from the drugs (Science, 15 March 2019, p. 1166).
Researchers have tried to reduce the heart risks by, for example, packaging the drugs in fat so they will home in on tumors, with limited success. But chemist Jacques Neefjes and his team at Leiden University and collaborators tried a different approach based on a surprising finding about how the drugs fight cancer, which they and a separate U.S. group reported in 2013. The textbook explanation is that the drugs kill rapidly dividing cells, such as those in a tumor, by blocking an enzyme they need to untangle and repair DNA as they replicate. But the researchers found doxorubicin also kills cancer cells by dislodging histones, the spherical proteins that DNA coils around like a spool to form a structure known as chromatin. This chromatin damage apparently interferes with the transcription of genes into proteins and other cell processes, Neefjes says.
In the new work, the Leiden team tested two anthracycline variants that remove histones without breaking DNA: an approved cancer drug called aclarubicin, and a tweaked version of doxorubicin they call diMe-Doxo. The compounds worked as well as the original drug, if not better, at killing cultured cancer cells and were nearly as effective at slowing tumor growth in mice. Yet mice prone to developing tumors that were dosed with aclarubicin did not show signs of heart damage, suggesting people treated with the drugs might be spared these effects. These mice were also much less likely to develop tumors later, the Leiden team reported online 17 June in the Proceedings of the National Academy of Sciences.
Uncoupling DNA damage from chromatin damage to detoxify doxorubicin
Xiaohang Qiao, Sabina Y. van der Zanden, Dennis P. A. Wander, Daniel M. Borràs, -Ying Song, Li, Suzanne van Duikeren, Noortje van Gils, Arjo Rutten, Tessa van Herwaarden, Olaf van Tellingen, Elisa Giacomelli, Milena Bellin, Valeria Orlova, Leon G. J. Tertoolen, Sophie Gerhardt, Jimmy J. Akkermans, Jeroen M. Bakker, Charlotte L. Zuur, Baoxu Pang, Anke M. Smits, Christine L. Mummery, Linda Smit, Arens, Li, Hermen S. Overkleeft, and Jacques Neefjes PNAS June 30, 2020 117 (26) 15182-15192;
Significance
Anthracyclines like doxorubicin are anticancer drugs, used by over 1 million cancer patients annually. However, they cause severe side effects, most notably, cardiotoxicity and therapy-related malignancies. It is unclear whether these side effects are directly linked to their anticancer activity. Doxorubicin exerts two activities: DNA damage and chromatin damage. Here, we show that both activities conspire in the cardiotoxicity, while doxorubicin variants with only chromatin-damaging activity remain active anticancer drugs devoid of side effects. This challenges the concept that doxorubicin works primarily by inducing DNA double-strand breaks and reveals another major anticancer activity, chromatin damage. Translating these observations will yield anticancer drugs for patients that are currently excluded from doxorubicin treatment and improve the quality of life of cancer survivors.
Abstract
The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.
Evaluation of the DNA- and chromatin-damaging activities of anthracyclines. (A) Structures of Topo II poisons used in this study, with the critical amine group in red. (B) K562 cells were treated for 2 h with 10 µM indicated drug. γH2AX levels were examined by Western blot. (C) Quantification of the γH2AX signal normalized to actin. (D) DSBs were analyzed by CFGE. (E) Quantification of relative broken DNA in D. (F) Part of the nucleus from MelJuSo-PAGFP-H2A cells was photoactivated. Photoactivated PAGFP-H2A was monitored by time-lapse confocal microscopy for 1 h in the absence or presence of indicated drug at 10 µM. Lines in Pre column define the regions of cytoplasm (C), nucleus (N), and activated area (A). (Scale bar, 10 µm.) (G) Quantification of the release of fluorescent PAGFP-H2A from the photoactivated region after drug administration. Two-way ANOVA, ****P < 0.0001. (H) Endogenously tagged scarlet-H2B U2OS cells were treated with 10 µM indicated drugs. Cells were fractionated, and the nuclear versus cytosolic fraction of H2B was examined by Western blot. Calnexin was used as cytosolic, and lamin A/C was used as nuclear marker. (I) The fraction of cytosolic versus nuclear H2B upon histone eviction by the drugs indicated is plotted. Two-way ANOVA, ***P < 0.001; ****P < 0.0001; ns, not significant. (J) Cell viability in K562 cells. Two-way ANOVA, Amr vs. Doxo, diMe-Doxo or Acla, **P < 0.01. (K) Relative IC50 values of each drug compared to Doxo in different cell lines. From Xiaohang Qiao et al. Uncoupling DNA damage from chromatin damage to detoxify doxorubicin. (2020); Proceedings of the National Academy of Sciences Jun 2020, 117 (26) 15182-15192; DOI: 10.1073/pnas.1922072117.
See Supplemental Videos of cardiac toxicity profile in mice of Doxo, diMe-Doxo or Acla
Download Movie_S03 (MP4) – Assessment of drug toxicity by echocardiography. Wild-type FVB mice were i.v. injected with the indicated drugs (Doxo, diMe-Doxo or Acla) for 8 times every week. Echocardiography was performed 12 weeks post start of the treatment. 3D reconstructions are shown of the heart in diastole of mice treated with the indicated drugs with the left ventricle in cyan and the left atrium in magenta. Related to Figure 4.
Download Movie_S04 (MP4) – Drug toxicity on hiPSC-derived cardiac microtissues. hiPSC-derived cardiac microtissues were stimulated at 1Hz, and velocity of microtissue-contraction was analysed after 24 hours exposure to indicated drugs (Doxo, diMe-Doxo or Acla). The Horn-Schunck Vector Flow analysis method was used to detect changes in pixel displacements during contraction of the microtissues in 3D. Related to Figure 4.
Anthracyclines are a class of antibiotics derived from certain types of Streptomyces bacteria. Anthracyclines are often used as cancer therapeutics and function in part as nucleic acid intercalating agents and inhibitors of the DNA repair enzyme topoisomerase II, thereby damaging nucleic acids in cancer cells, preventing the cells from replicating. One example of an anthracycline cancer therapeutic is doxorubicin, which is used to treat a variety of cancers including breast cancer, lung cancer, ovarian cancer, lymphoma, and leukemia. The 6-maleimidocaproyl hydrazone of doxorubicin (DOXO-EMCH) was originally synthesized to provide an acid-sensitive linker that could be used to prepare immunoconjugates of doxorubicin and monoclonal antibodies directed against tumor antigens (Willner et al., Bioconjugate Chem 4:521-527 (1993)). In this context, antibody disulfide bonds are reduced with dithiothreitol to form free thiol groups, which in turn react with the maleimide group of DOXO-EMCH to form a stable thioether bond. When administered, the doxorubicin-antibody conjugate is targeted to tumors containing the antigen recognized by the antibody. Following antigen-antibody binding, the conjugate is internalized within the tumor cell and transported to lysosomes. In the acidic lysosomal environment, doxorubicin is released from the conjugate intracellularly by hydrolysis of the acid-sensitive hydrazone linker. Upon release, the doxorubicin reaches the cell nucleus and is able to kill the tumor cell. For additional description of doxorubicin and
DOXO-EMCH see, for example, U.S. Patents 7,387,771 and 7,902,144 and U.S. Patent Application No. 12/619,161, each of which are incorporated in their entirety herein by reference.
[0003] A subsequent use of DOXO-EMCH was developed by reacting the molecule in vitro with the free thiol group (Cys-34) on human serum albumin (HSA) to form a stable thioether conjugate with this circulating protein (Kratz et al, J Med Chem 45:5523-5533 (2002)). Based on these results, it was
hypothesized that intravenously-administered DOXO-EMCH would rapidly conjugate to HSA in vivo and that this macromolecular conjugate would preferentially accumulate in tumors due to an “enhanced permeability and retention” (EPR) intratumor effect (Maeda et al., J Control Release 65:271-284 (2000)).
[0004] Acute and repeat-dose toxicology studies with DOXO-EMCH in mice, rats, and dogs identified no toxicity beyond that associated with doxorubicin, and showed that all three species had significantly higher tolerance for DOXO-EMCH compared to doxorubicin (Kratz et al, Hum Exp Toxicol 26: 19-35 (2007)). Based on the favorable toxicology profile and positive results from animal tumor models, a Phase 1 clinical trial of DOXO-EMCH was conducted in 41 advanced cancer patients (Unger et al, Clin Cancer Res 13:4858-4866 (2007)). This trial found DOXO-EMCH to be safe for clinical use. In some cases, DOXO-EMCH induced tumor regression.
[0005] Due to the sensitivity of the acid-cleavable linker in DOXO-EMCH, it is desirable to have formulations that are stable in long-term storage and during reconstitution (of, e.g., previously lyophilized compositions) and administration. DOXO-EMCH, when present in compositions, diluents and administration fluids used in current formulations, is stable only when kept at low temperatures. The need to maintain DOXO-EMCH at such temperatures presents a major problem in that it forces physicians to administer cold (4°C) DOXO-EMCH compositions to patients. Maintaining DOXO-EMCH at low temperatures complicates its administration in that it requires DOXO-EMCH to be kept at 4°C and diluted at 4°C to prevent degradation that would render it unsuitable for patient use. Further, administration at 4°C can be harmful to patients whose body temperature is significantly higher (37°C).
[0006] Lyophilization has been used to provide a stable formulation for many drugs. However, reconstitution of lyophilized DOXO-EMCH in a liquid that does not maintain stability at room temperature can result in rapid decomposition of DOXO-EMCH. Use of an inappropriate diluent to produce an injectable composition of DOXO-EMCH can lead to decreased stability and/or solubility. This decreased stability manifests itself in the cleavage of the linker between the doxorubicin and EMCH moieties, resulting in degradation of the DOXO-EMCH into two components: doxorubicin and linker-maleimide. Thus, stable,
reconstituted lyophilized solutions of anthracycline-EMCH (e.g., DOXO-EMCH), and injectable compositions containing the same, are required to solve these problems and to provide a suitable administration vehicle that can be used reasonably in treating patients both for clinical trials and commercially.
DOXO-EMCH. The term “DOXO-EMCH,” alone or in combination with any other term, refers to a compound as depicted by the following structure:
OH
DOXO-EMCH is also referred to as (E)-N’-(l-((2S,4S)-4-(4-amino-5-hydroxy-6- methyl-tetrahydro-2H-pyran-2-yloxy-2,5 , 12-trihydroxy-7-methoxy-6, 11- dioxol,2,3,4,6,l l-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2H- pyrrol- 1 (5H)yl)hexanehydrazide»HCl.
DOXIL (doxorubicin HCl liposome injection) is doxorubicin hydrochloride (HCl) encapsulated in STEALTH® liposomes for intravenous administration.
Doxorubicin is an anthracycline topoisomerase inhibitor isolated from Streptomyces peucetius var. caesius.
Doxorubicin HCl, which is the established name for (8S,10S)-10-[(3-amino-2,3,6-trideoxyα- L-lyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy5,12- naphthacenedione hydrochloride, has the following structure:
The molecular formula of the drug is C27H29NO11•HCl; its molecular weight is 579.99.
DOXIL (doxorubicin hcl liposome injection) is provided as a sterile, translucent, red liposomal dispersion in 10-mL or 30-Ml glass, single use vials. Each vial contains 20 mg or 50 mg doxorubicin HCl at a concentration of 2 mg/mL and a pH of 6.5. The STEALTH® liposome carriers are composed of N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero3- phosphoethanolamine sodium salt (MPEG-DSPE), 3.19 mg/mL; fully hydrogenated soy phosphatidylcholine (HSPC), 9.58 mg/mL; and cholesterol, 3.19 mg/mL. Each mL also contains ammonium sulfate, approximately 2 mg; histidine as a buffer; hydrochloric acid and/or sodium hydroxide for pH control; and sucrose to maintain isotonicity. Greater than 90% of the drug is encapsulated in the STEALTH® liposomes
MONDAY Feb. 4, 2013 — The first generic version of the cancer drug Doxil (doxorubicin hydrochloride liposome injection) has been approved by the U.S. Food and Drug Administration, which says the action should help relieve shortages of the brand-name medication.
Doxil is on the agency’s drug shortage list. The list empowers the FDA’s Office of Generic Drugs to grant priority review to generic equivalents, the agency said Monday in a news release.
Noting that generics were of the same quality and strength as the original drugs, the FDA said: “Generic manufacturing and packaging sites must pass the same quality standards as those of brand-name drugs.”
Generic Doxil will be produced by Sun Pharma Global FZE in 20 milligram and 50 milligram vials.
CARDIOMYOPATHY, SECONDARY MALIGNANCIES, EXTRAVASATION AND TISSUE NECROSIS, and SEVERE MYELOSUPPRESSION
Cardiomyopathy: Myocardial damage can occur with doxorubicin with incidences from 1% to 20% for cumulative doses from 300 mg/m² to 500 mg/m² when doxorubicin is administered every 3 weeks. The risk of cardiomyopathy is further increased with concomitant cardiotoxic therapy. Assess left ventricular ejection fraction (LVEF) before and regularly during and after treatment with doxorubicin.
Secondary Malignancies : Secondary acute myelogenous leukemia (AML) and myelodys plastic syndrome (MDS) occur at a higher incidence in patients treated with anthracyclines, including doxorubicin.
Extravasation and Tissue Necrosis : Extravasation of doxorubicin can result in severe local tissue injury and necrosis requiring wide excision and skin grafting. Immediately terminate the drug, and apply ice to the affected area.
Severe myelosuppression resulting in serious infection, septic shock, requirement for trans fusions, hospitalization, and death may occur.
The history of this finding is quite interesting and, as I remember in a talk given by Dr. Sood in mid-2000’s, a microarray conducted by his lab had showed overexpression of the β2-AR (β2 adrenergic receptor in ovarian cancer cells relative to normal epithelium. At the time it appeared an interesting result however most of the cancer (and ovarian cancer) field were concentrating on the tyrosine kinase signaling pathways as potential therapeutic targets, as much promising translational research in this area was in focus at the time. As a result of this finding and noticing that sustained β-adrenergic stimulation can promote ovarian cancer cell growth (Sood, 2006), Dr. Sood’s group have been studying the effects of β-adrenergic signaling om ovarian cancer. In addition it has been shown that propanalol can block VEGF signaling and norepinephrine increased MMP2 and MMP9 expression, an effect mediated by the β2-AR.
The above re-post of a Scoop-IT describes promising results of a clinical trial for use of selective beta blockers in ovarian cancer. As to date, there have been many clinical trials initiated in ovarian cancer and most have not met with success for example the following posts:
which contains an interview with Dr. Maurie Markman (Vice President, Patient Oncology Services, and National Director for Medical Oncology, Cancer Treatment Centers of America) and Dr. Kathy D. Miller, Indiana University School of Medicine) and discusses how each patient’s ovarian cancer is genetically unique and needs to be treated as such
Therefore the mainstay therapy is still carboplatin plus a taxane (Taxotere, Abraxane). The results of this clinical trial show a 5 month improvement in survival, which for a deadly disease like ovarian cancer is a significant improvement.
First below is a SUMMARY of the paper’s methodology and findings.
Methods:
Four participating institutions collected retrospective patient data and pathology reports from 1425 patients diagnosed with epithelial ovarian cancer (EOC)
Medical records were evaluated for use of both selective and nonselective β-blockers
β-blockers were used for various indications however most common indication was treatment for hypertension (71% had used β1 selective blockers while rest of patients taking β blockers were given nonselective blockers for a host of other indications)
most patients had stage III/IV disease and in general older (median age 63 years)
The authors looked at overall survival (OS) however progression free survival PFS) was not calculated
Results:
Hypertension was associated with decreased survival (40.1 monts versus 47.4 months for normotensive patients)
Overall Survival for patients on any β blockers was 47.8 months versus 42.0 months for nonusers
Patients receiving nonselective β blockers has an OS of 94.9 months versus 38 months for EOC patients receiving β1-selective blockers
No effect of diabetes mellitus on survival
Authors Note on Limitations of Study:
Retrospective in nature
Lack of documentation of dosage, trade-name and duration of β-blocker use
Important to stratify patients on selectivity of β-blocker since Eskander et. al. found no difference of Progression Free Survival and non-selective β-blocker
Several β adrenergic receptor polymorphisms may exist and no downstream biomarker evaluated to determine effect on signaling; could it be a noncanonical effect?
The goal of this brief, added curation is to paint a historical picture, and highlight the scientific findings which led up to the rationale behind this clinical trial.
How the βeta Adrenergic Receptor (βAR) Became a Target for Ovarian Cancer
.
A. βAR and its signaling over-expressed in ovarian cancer
Lee JW, Shahzad MM, Lin YG, Armaiz-Pena G, Mangala LS, Han HD, Kim HS, Nam EJ, Jennings NB, Halder J, Nick AM, Stone RL, Lu C, Lutgendorf SK, Cole SW, Lokshin AE, Sood AK.
Sood group wanted to mimic the surgical stress after laparoscopic surgery to see if surgical stress would promote the growth of micrometasteses remaining after surgical tumor removal. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for beta-adrenergic receptor signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and vascular endothelial growth factor expression, which were blocked by propranolol treatment. Tumor growth after surgery was decreased in a mouse null for βAR. Levels of cytokines G-CSF, IL-1a, IL-6, and IL-15were increased after surgery
Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis J Biol Chem. 2010 Nov 12;285(46):35462-70. doi: 10.1074/jbc.M110.109579. Epub 2010 Sep 8.
A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.
Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer
Requests for reprints:
Anil K. Sood, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M. D. Anderson Cancer Center, 1155 Herman Pressler, CPB6.3244, Unit 1362, Houston, TX 77230-1439. Phone: 713-745-5266; Fax: 713-792-7586; E-mail: asood@mdanderson.org.
Abstract
There is growing evidence that chronic stress and other behavioral conditions are associated with cancer pathogenesis and progression, but the mechanisms involved in this association are poorly understood. We examined the effects of two mediators of stress, norepinephrine and epinephrine, on the activation of signal transducer and activator of transcription-3 (STAT3), a transcription factor that contributes to many promalignant pathways. Exposure of ovarian cancer cell lines to increasing concentrations of norepinephrine or epinephrine showed that both independently increased levels of phosphorylated STAT3 in a dose-dependent fashion. Immunolocalization and ELISA of nuclear extracts confirmed increased nuclear STAT3 in response to norepinephrine. Activation of STAT3 was inhibited by blockade of the β1- and β2-adrenergic receptors with propranolol, and by blocking protein kinase A with KT5720, but not with the α receptor blockers prazosin (α1) and/or yohimbine (α2). Catecholamine-mediated STAT3 activation was not inhibited by pretreatment with an anti–interleukin 6 (IL-6) antibody or with small interfering RNA (siRNA)–mediated decrease in IL-6 or gp130. Regarding the effects of STAT3 activation, exposure to norepinephrine resulted in an increase in invasion and matrix metalloproteinase (MMP-2 and MMP-9) production. These effects were completely blocked by STAT3-targeting siRNA. In mice, treatment with liposome-incorporated siRNA directed against STAT3 significantly reduced isoproterenol-stimulated tumor growth. These studies show IL-6–independent activation of STAT3 by norepinephrine and epinephrine, proceeding through the β1/β2-adrenergic receptors and protein kinase A, resulting in increased matrix metalloproteinase production, invasion, and in vivo tumor growth, which can be ameliorated by the down-regulation of STAT3. [Cancer Res 2007;67(21):10389–96]
Objective: Stress hormones such as catecholamines can augment tumor metastasis and angiogenesis; however, the prevalence and clinical significance of adrenergic receptors in human ovarian cancer is unknown and is the focus of the current study. Methods: After IRB approval, paraffin-embedded samples from 137 patients with invasive epithelial ovarian carcinoma were examined for \#946;1- and \#946;2-adrenergic receptor (ADRB1 and ADRB2, respectively) expression. Correlations with clinical outcomes were determined using parametric and non-parametric tests. Survival analyses were performed using the Kaplan-Meier method. Expression of ADRB1 and -2 was examined by quantitative RT-PCR in 15 freshly extracted human ovarian carcinoma cells. Human ovarian carcinoma cells then underwent time-variable adrenergic stimulation, and tumorigenic and angiogenic cytokine levels were examined by ELISA. Results: Sixty-six percent of the tumors had high expression of ADRB1; 80% of specimens highly expressed ADRB2. Univariate analyses demonstrated that high ADRB1 expression was associated with serous histology (p=0.03) and the presence of ascites (p=0.03), while high expression of ADRB2 was associated with advanced stage (p=0.008). Moreover, high ADRB2 expression was associated with the lower overall survival (2.2 vs. 6.5 years; p<0.001). In multivariate analysis, controlling for FIGO stage, grade, cytoreduction, age, and ADRB expression, only FIGO stage, cytoreduction status, age, and ADRB status retained statistical significance in predicting overall survival. In tumor cells freshly isolated from human ovarian cancers, 75% of samples had high expression of ADRB2 while most lacked ADRB1 compared to normal surface epithelium. Stimulation of the freshly isolated ADRB2-positive human ovarian cancer cells with norepinephrine resulted in increased levels of cAMP and increased angiogenic cytokines IL-6 and VEGF. Conclusions:ADRB2 are frequently found on human ovarian tumors and are strongly associated with poor clinical outcome. These findings support a direct mechanism by which stress hormones modulate ovarian cancer growth and metastasis as well as provide a basis for therapeutic targeting.
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA
Abstract
Purpose: Catecholamine mediated stress effects are known to induce production of various pro-inflammatory cytokines. However, the mechanism and functional effect of adrenergic signaling in driving inflammation via pro-inflammatory metabolites is currently unknown. Here we address the functional and biological consequences of adrenergic-induced Cox2/PGE2 axis activation in ovarian cancer metastasis.
Methods: We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite PGE2 in vitro and in vivo. To study tumor-specific effects on catecholamine-derived expression of PTGS2, we used a novel DOPC delivery system of PTGS2 siRNA.
Results: Our results revealed that levels of PGs were significantly increased in patients with high depressive scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression and protein levels in Skov3 and HeyA8. PGE2 ELISA confirmed that upon treatment with NE, PGE2 levels were increased in conditioned medium from Skov3 and HeyA8 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES as well as PGE2 levels in supernatant. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. ChIP analysis showed enrichment of Nf-kB binding to the promoter region of PTGS2 and PTGES by 2.4 and 4.0 fold respectively when Skov3ip1 cells were treated with NE. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 cells in the presence of NE. Importantly, in the Skov3-ip1 restraint stress orthotopic model, silencing PTGS2 abrogated stress mediated effects and decreased tumor burden by 70% compared to control siRNA with restraint stress.
Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu mediated by prostaglandins that drives tumor progression and metastasis in ovarian cancer.
Citation Format: Archana S. Nagaraja, Piotr Dorniak, Nouara Sadaoui, Guillermo Armaiz-Pena, Behrouz Zand, Sherry Y. Wu, Julie K. Allen, Rajesha Rupaimoole, Cristian Rodriguez-Aguayo, Sunila Pradeep, Lin Tan, Rebecca A. Previs, Jean M. Hansen, Peiying Yang, Garbiel Lopez-Berestein, Susan K. Lutgendorf, Steve Cole, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3368. doi:10.1158/1538-7445.AM2015-3368
Other Article in This Open Access Journal on Ovarian Cancer Include
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.
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
Increased DNA Repair Mechanisms of Platinated Lesion Lead to ChemoResistance
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
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.
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.
Multiple Platinum Analogs of Cisplatin (like Oxaliplatin )Had Been Designed to be Sensitive in MMR Deficient Tumors
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
Johnson, S. W. et al. Relationship between platinum-DNA adduct formation, removal, and cytotoxicity in cisplatin sensitive and resistant human ovarian cancer cells. Cancer Res54, 5911-5916 (1994).
Eastman, A. The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacology and Therapeutics34, 155-166 (1987).
Zhen, W. et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Molecular and Cellular Biology12, 3689-3698 (1992).
Durocher, D. & Jackson, S. P. DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol13, 225-231 (2001).
el-Deiry, W. S. p21/p53, cellular growth control and genomic integrity. Curr Top Microbiol Immunol227, 121-37 (1998).
Ewen, M. E. & Miller, S. J. p53 and translational control. Biochim Biophys Acta1242, 181-4 (1996).
Gartel, A. L., Serfas, M. S. & Tyner, A. L. p21–negative regulator of the cell cycle. Proc Soc Exp Biol Med213, 138-49 (1996).
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. Oncogene19, 2165-70 (2000).
Davies, N. P., Hardman, L. C. & Murray, V. The effect of chromatin structure on cisplatin damage in intact human cells. Nucleic Acids Res28, 2954-2958 (2000).
Vichi, P. et al. Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP. Embo J16, 7444-7456 (1997).
Xiao, G. et al. A DNA damage signal is required for p53 to activate gadd45. Cancer Res60, 1711-9 (2000).
Other articles in this Open Access Journal on ChemoResistance Include:
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:
The Changing Views How Bone Remodeling Occurs
Early Development of Agents that Alter Bone Remodeling and Early Use in Cancer Patients
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
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.
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:
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
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
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.
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).
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.
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:
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 thisLINK 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.
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 animalsmimicking 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.
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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