Feeds:
Posts
Comments

Posts Tagged ‘ubiquitination’


Curator: Ritu Saxena, Ph.D.

Melanoma

Melanoma represents approximately 4% of human skin cancers, yet accounts for approximately 80% of deaths from cutaneous neoplasms. It remains one of the most common types of cancer among young adults. Melanoma is recognized as the most common fatal skin cancer with its incidence rising to 15 fold in the past 40 years in the United States. Melanoma develops from the malignant transformation of melanocytes, the pigment-producing cells that reside in the basal epidermal layer in human skin. (Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36 ; Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6).  Classic clinical signs of melanoma include change in color, recent enlargement, nodularity, irregular borders, and bleeding. Cardinal signs of melanoma are sometimes referred to by the mnemonic ABCDEs (asymmetry, border irregularity, color, diameter, elevation) (Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824).

Clinical characteristics

Melanoma primarily affects fair-haired and fair-skinned individuals, and those who burn easily or have a history of severe sunburn are at higher risk than their darkly pigmented, age-matched controls. The exact mechanism and wavelengths of UV light that are the most critical remain controversial, but both UV-A (wavelength 320–400 nm) and UV-B (290–320 nm) have been implicated (Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112). Case-control studies have identified several risk factors in populations susceptible to developing melanoma. MacKie RM et al (1989) stated that the relative risk of cutaneous melanoma is estimated from the four strongest risk factors identified by conditional logistic regression. These factors are

  • total number of benign pigmented naevi above 2 mm diameter;
  • freckling tendency;
  • number of clinically atypical naevi (over 5 mm diameter and having an irregular edge, irregular pigmentation, or inflammation); and
  • a history of severe sunburn at any time in life.

Use of this risk-factor chart should enable preventive advice for and surveillance of those at greatest risk (MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90).

Cutaneous melanoma can be subdivided into several subtypes, primarily based on anatomic location and patterns of growth (Table 1).

Image

Table 1: Clinical Classification of Melanoma (Chudnovsky Y, et al, 2005)

The genetics of melanoma

As in many cancers, both genetic predisposition and exposure to environmental agents are risk factors for melanoma development. Many studies conducted over several decades on benign and malignant melanocytic lesions as well as melanoma cell lines have implicated numerous genes in melanoma development and progression.

Image

Table 2: Genes involved in Melanoma (Chudnovsky Y, et al, 2005)

Apart from the risk factors such as skin pigmentation, freckling, and so on, another significant risk factor is ‘strong family history of melanoma’. Older case-control studies of patients with familial atypical mole-melanoma (FAMM) syndrome suggested an elevated risk of ∼434-to 1000-fold over the general population (Greene MH, et al, Ann Intern Med, Apr 1985;102(4):458-65). A more recent meta-analysis of family history found that the presence of at least one first-degree relative with melanoma increases the risk by 2.24-fold (Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59). Genetic studies of melanoma-prone families have given important clues regarding melanoma susceptibility loci.

CDKN2A, the familial melanoma locus

CDKN2A is located at chromosome 9p21 and is composed of 4 exons (E) – 1α, 1β, 2, and 3. LOH or mutations at this locus cosegregated with melanoma susceptibility in familial melanoma kindred and 9p21 mutations have been observed in different cancer cell lines. The locus encodes two tumor suppressors via alternate reading frames, INK4 (p16INK4a) and ARF (p14ARF). INK4A and ARF encode alternative first exons, 1α and 1β respectively and different promoters. INK4A is translated from the splice product of E1α, E2, and E3, while ARF is translated from the splice product of E1β, E2, and E3. Second exons of the two proteins are shared and two translated proteins share no amino acid homology.

INK4A is the founding member of the INK4 (Inhibitor of cyclin-dependent kinase 4) family of proteins and inhibits the G1 cyclin-dependent kinases (CDKs) 4/6, which phosphorylate and inactivate the retinoblastoma protein (RB), thereby allowing for S-phase entry. Thus, loss of INK4K function promotes RB inactivation through hyperphosphorylation, resulting in unconstrained cell cycle progression.

ARF (Alternative Reading Frame) protein of the locus inhibits HDM2-mediated ubiquitination and subsequent degradation of p53. Thus, loss of ARF inactivates another tumor suppressor, p53. The loss of p53 impairs mechanisms that normally target genetically damaged cells for cell cycle arrest and/or apoptosis, which leads to proliferation of damaged cells. Loss of CDKN2A therefore contributes to tumorigenesis by disruption of both the pRB and p53 pathways.

figure 1

Figure 1:  Genetic encoding and mechanism of action of INK4A and ARF.

(Chudnovsky Y, et al, 2005)

RAF and RAS pathways

A genetic hallmark of melanoma is the presence of activating mutations in the oncogenes BRAF and NRAS, which are present in 70% and 15% of melanomas, respectively, and lead to constitutive activation of mitogen-activated protein kinase (MAPK) pathway signaling. However, molecules that inhibit MAPK pathway–associated kinases, like BRAF and MEK, have shown only limited efficacy in the treatment of metastatic melanoma. Thus, a deeper understanding of the cross talk between signaling networks and the complexity of melanoma progression should lead to more effective therapy.

NRAS mutations activate both effector pathways, Raf-MEK-ERK and PI3K-Akt in melanoma. The Raf-MEK-ERK pathway may also be activated via mutations in the BRAF gene. In a subset of melanomas, the ERK kinases have been shown to be constitutively active even in the absence of NRAS or BRAF mutations. The PI3K-Akt pathway may be activated through loss or mutation of the tumor suppressor gene PTEN, occurring in 30–50% of melanomas, or through gene amplification of the AKT3 isoform. Activation of ERK and/or Akt3 promotes the development of melanoma by various mechanisms, including stimulation of cell proliferation and enhanced resistance to apoptosis.

JCI0524808.f3

Figure 2: Schematic of the canonical Ras effector pathways Raf-MEK-ERK and PI3K-Akt in melanoma.

Curtin et al (2005) compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and NRAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. Significant differences were observed in number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Eighty-one percent of the melanomas on skin without sun-induced damaged had mutations in BRAF or NRAS. Melanomas with wild-type BRAF or NRAS frequently had increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), downstream components of the RAS-BRAF pathway. Thus, the genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or NRAS. (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47).

Genetic Heterogeneity of Melanoma

Melanoma exhibits molecular heterogeneity with markedly distinct biological and clinical behaviors. Lentigo maligna melanomas, for example, are indolent tumors that develop over decades on chronically sun-exposed area such as the face. Acral lentigenous melanoma, or the other hand, develops on sun-protected regions, tend to be more aggressive. Also, transcription profiling has provided distinct molecular subclasses of melanoma. It is also speculated that alterations at the DNA and RNA and the non-random nature of chromosomal aberrations may segregate melanoma tumors into subtypes with distinct clinical behaviors.

The melanoma gene atlas

Whole-genome screening technologies such as spectral karyotype analysis and array-CGH have identified many recurrent nonrandom chromosomal structural alterations, particularly in chromosomes 1, 6, 7, 9, 10, and 11 (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47); however, in most cases, no known or validated targets have been linked to these alterations.

In A systematic high-resolution genomic analysis of melanocytic genomes, array-CGH profiles of 120 melanocytic lesions, including 32 melanoma cell lines, 10 benign melanocytic nevi, and 78 melanomas (primary and metastatic) by Chin et al (2006) revealed a level of genomic complexity not previously appreciated. In total, 435 distinct copy number aberrations (CNAs) were defined among the metastatic lesions, including 163 recurrent, high-amplitude events. These include all previously described large and focal events (e.g., 1q gain, 6p gain/6q loss, 7 gain, 9p loss, and 10 loss). Genomic complexity observed in primary and benign nevi melanoma is significantly less than that observed in metastatic melanoma (Figure 3)  (Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182).

Genetic heterogeneity Melanoma

Figure 3: Genome comparisons of melanocyte lesions (Chin L, et al, 2006)

Thus, genomic profiling of various melanoma progression types could reveal important information regarding genetic events those likely drive as metastasis and possibly, reveal provide cues regarding therapy targeted against melanoma.

Reference:

  1. Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36
  2. Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6
  3. Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824
  4. Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112
  5. MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90)
  6. Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59)
  7. Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47
  8. Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182

Related articles on Melanoma on this Open Access Online Scientific Journal, include the following: 

Thymosin alpha1 and melanoma Author/Editor- Tilda Barliya, Ph.D.

A New Therapy for Melanoma Reporter- Larry H Bernstein, M.D.

Melanoma: Molecule in Immune System Could Help Treat Dangerous Skin Cancer Reporter: Prabodh Kandala, Ph.D.

Why Braf inhibitors fail to treat melanoma. Reporter: Prabodh Kandala, Ph.D.

Read Full Post »


Author and Curator: Ritu Saxena, Ph.D.

Introduction: Mitochondrial fission & fusion

Mitochondria, double membranous and semi-autonomous organelles, are known to convert energy into forms that are usable to the cell. Apart from being sites of cellular respiration, multiple roles of mitochondria have been emphasized in processes such as cell division, growth and cell death. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. Mitochondria have been discussed in several posts published in the Pharmaceutical Intelligence blog.

Mitochondria do not exist as lone organelles, but are part of a dynamic network that continuously undergoes fusion and fission in response to various metabolic and environmental stimuli. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid. Mitochondrial fusion and fission within a cell is speculated to be involved in several functions including mtDNA DNA protection, alteration of cellular energetics, and regulation of cell division.

Proteins involved in mitochondrial fission & fusion

Multiple mitochondrial membrane GTPases that regulate mitochondrial networking have recently been identified. They are classified as fission and fusion proteins:

Fusion proteins: Members of dynamin family of protein, mitofusin 1 (Mfn-1) and mitofusin 2 (Mfn-2), are involved in fusion between mitochondria by tethering adjacent mitochondria. These proteins have two transmembrane segments that anchor them in the mitochondrial outer membrane. Mutations in Mitofusin proteins gives rise to fragmented mitochondria, but this can be reversed by mutations in mammalian Drp1. Mitochondrial inner membranes are fused by dynamin family members called Opa1.

Fission proteins: Another member of the dynamin family of proteins, dynamin-related protein 1 (Drp-1) mediates fission of mitochondria. Drp-1 is activated by phosphorylation. Drp-1 proteins are largely cytosolic, but cycle on and off of mitochondria as needed for fission. Fission is a complex process and involves a series of well-defined stages and proteins. Cytosolic Drp-1 is activated by calcineurin or other cytosolic signaling proteins after which it translocates to the mitochondrial tubules where it assembles into foci through its interaction with another protein, hFis1. Once Drp-1 rings assemble on the constricted spots, outer membrane of mitochondria undergoes fission through GTP hydrolysis. Drp-1 is now left bound to one of the newly formed mitochondrial ends after which it slowly disassembles before returning to the cytoplasm.

Control of mitochondrial fission & fusion

  • Mitochondrial fission and fusion are controlled by several regulatory mechanisms. Few of which are mentioned as follows:
  • Drp-1 activation by Cdk1/Cyclin B mediated phosphorylation during mitosis – triggers fission
  • Drp-1 inactivation by cAMP-dependent protein kinase (PKA) in quiescent cells- prevents fission
  • Drp-1 activation after reversal of PKA phosphorylation by Calcineurin- triggers fission
  • Ubiquination of fission and fusion proteins by E3 ubiquitin ligase- alters fission
  • Sumoylation of fission proteins – regulates fission

Imparied mitochondrial fission leads to loss of mtDNA

Mitochondrial fission plays an important role in mitochondrial and cellular homeostasis. It was reported by Parone et al (2008) that preventing mitochondrial fission by down-regulating expression of Drp-1 lead to loss of mtDNA and mitochondrial dysfunction. An increase in cellular reactive oxygen species (ROS) was observed. Other cellular implications included depletion of cellular ATP, inhibition of cell proliferation and autophagy. The observations were made in HeLa cells.

MicroRNA regulation of mitochondrial fission

Although several factors have been attributed to the regulation of mitochondrial fission, the mechanism still remains poorly understood. Recently, regulation of mitochondrial fission via miRNAs has become a topic of interest. Following miRNAs have been found to be involved in mitochondrial fission:

  • miR-484:  Wang et al (2012) demonstrated that miR-484 was able to regulate mitochondrial fission by suppressing the translation of a fission protein Fis1, leading to inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. The authors showed that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. Underlying mechanism involved transactivation of miR-484 by a transcription factor, Foxo3a and miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation.
  • miR-499: miR-499 was reported by Wang et al (2011) to be able to directly target both the α- and β-isoforms of the calcineurin catalytic subunit. Suppression of calcineurin-mediated dephosphorylation of  Drp-1 lead to inhibition of the fission machinery ultimately resulting in the inhibition of cardiomyocyte apoptosis. miR-499 levels, by altering mitochondrial fusion were able affect the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. Modulation of miR-499 expression could provide a therapeutic approach for myocardial infarction treatment.
  • miR-30: It was reported by Li et al (2010) that miR-30 family members were able to inhibit mitochondrial fission and also the resulting apoptosis. While exploring the underlying molecular mechanism, the authors identified that miR-30 family members can suppress p53 expression. When cell received apoptotic stimulation, p53 was found to transcriptionally activate the fission protein, Drp-1. Drp-1 was able to induce mitochondrial fission. miR-30 family members were observed to inhibit mitochondrial fission through attenuation of p53 expression and its downstream target Drp-1.

Mitochondrial fission & fusion as a therapeutic target

Since alteration of mitochondrial fission and fusion have been reported to affect various cellular processes including apoptosis, proliferation, ATP consumption, the proteins involved in the process of fission and fusion might be harnessed as therapeutic target.

Mentioned below is a description of research where dynamics of the mitochondrial organelle has been utilized as a therapeutic target:

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

A recent article published by Rehman et al (2012) in the FASEB journal drew much attention after interesting observations were made in the mitochondria of lung adenocarcinoma cells. The mitochondrial network of these cells exhibited both impaired fusion and enhanced fission. It was also found that the fragmented phenotype in multiple lung adenocarcinoma cell lines was associated with both a down-regulation of the fusion protein, Mfn-2 and an upregulation of expression of fission protein, Drp-1. The imbalance of Drp-1/Mfn-2 expression in human lung cancer cell lines was reported to promote a state of mitochondrial fission. Similar increase in Drp-1 and decrease in Mfn-2 was observed in the tissue samples from patients compared to adjacent healthy lung. Authors used complementary approaches of Mfn-2 overexpression, Drp-1 inhibition, or Drp-1 knockdown and were able to observe reduction of cancer cell proliferation and an increase spontaneous apoptosis. Thus, the study identified mitochondrial fission and Drp-1 activation as a novel therapeutic target in lung cancer.

Image

Reference:

Research articles-

http://www.ncbi.nlm.nih.gov/pubmed/20556877

http://www.ncbi.nlm.nih.gov/pubmed?term=18806874

http://www.ncbi.nlm.nih.gov/pubmed/22510686

http://www.ncbi.nlm.nih.gov/pubmed/21186368

http://www.ncbi.nlm.nih.gov/pubmed?term=20062521

http://www.ncbi.nlm.nih.gov/pubmed?term=22321727

News brief:

http://www.uchospitals.edu/news/2012/20120221-mitochondria.html

http://news.uchicago.edu/article/2012/02/23/energy-network-within-cells-may-be-new-target-cancer-therapy

http://www.doctortipster.com/7881-mitochondria-could-represent-a-new-target-for-cancer-therapy-according-to-new-study.html

Related reading:

Reviewer: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Author and Curator: Larry H Bernstein, MD, FACP https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Reporter and Editor: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Author and Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/09/10/%CE%B2-integrin-emerges-as-an-important-player-in-mitochondrial-dysfunction-associated-gastric-cancer/

Author: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Author and Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

Reporter: Venkat S. Karra, PhD

https://pharmaceuticalintelligence.com/2012/08/14/detecting-potential-toxicity-in-mitochondria/

Reporter: Aviva Lev-Ari, PhD, RN https://pharmaceuticalintelligence.com/2012/08/01/mitochondrial-mechanisms-of-disease-in-diabetes-mellitus/

Author and Curator: Ritu Saxena, PhD; Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Read Full Post »