PhD candidate, University of Massachusetts Medical School, Worcester MA, USA
Understanding the mechanisms behind how a cancer starts is an important step towards finding a cure
A normal, diploid cell can progress through the cell cycle, divide flawlessly and duplicate its genome into resulting daughter cells. However, if this process occurs erroneously the subsequent daughter cells inherit an abnormal DNA content, a condition known as aneuploidy. In cancer, this process is commonly corrupted giving rise to increased aneuploidy and genomic instability within the tumor cells. Solid tumors frequently undergo whole genome duplications, leading to an intermediate tetraploid state. Tetraploidy precedes aneuploidy and is thought to be important in driving tumorigenesis, however the mechanisms driving tetraploidy in the first place, are unclear.
My research focuses on the role of tetraploidy in the progression of melanoma. Melanoma is the most lethal form of skin cancer, responsible for 75% of all skin cancer deaths and is associated with a high mortality rate due to its potential to invade and metastasize rapidly. In 2017, an estimated 87,110 new cases and 9,730 deaths are expected from melanoma in the United States alone. Melanoma is composed of aberrant melanocytes, the pigment producing cells of the skin. In order to gain insight into the early cellular events contributing to melanoma formation, we examined melanocytes in a melanoma-prone zebrafish model with an activated form of the commonly mutated human oncogene, BRAF. We found that melanocytes in these transgenic zebrafish were binucleate and tetraploid. In addition to activated BRAF, zebrafish lacking the common tumor suppressor, p53, exhibited a polyploid DNA content in their melanocytes and melanomas. These data implicate increased BRAF activity as a contributor to tetraploidy, and the lack of p53 in the progression of melanomas. We are currently using in vitro live cell imaging approaches to determine the mechanism by which these tetraploids arise and we are conducting retrospective bioinformatic analyses to determine whether these melanomas progress through tetraploid intermediates. This project aims to understand the earliest events in tumorigenesis and how common mutations are responsible for these events. It will also help us determine important mechanisms that cause genomic instability in human cancers, with hopes of ultimately finding a cure. I have designed this pinboard with to emphasize the significance of tetraploidy and genome instability in cancer by pinning some of the seminal papers in the field as well as exciting current literature. I hope you enjoy it!
Abstract: Human melanocytic nevi (moles) are benign lesions harboring activated oncogenes, including BRAF. Although this oncogene initially acts mitogenically, eventually, oncogene-induced senescence (OIS) ensues. Nevi can infrequently progress to melanomas, but the mechanistic relationship with OIS is unclear. We show here that PTEN depletion abrogates BRAF(V600E)-induced senescence in human fibroblasts and melanocytes. Correspondingly, in established murine BRAF(V600E)-driven nevi, acute shRNA-mediated depletion of PTEN prompted tumor progression. Furthermore, genetic analysis of laser-guided microdissected human contiguous nevus-melanoma specimens recurrently revealed identical mutations in BRAF or NRAS in adjacent benign and malignant melanocytes. The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Pharmacologic PI3K inhibition in melanoma cells suppressed proliferation and induced the senescence-associated tumor suppressor p15(INK4B). This treatment also eliminated subpopulations resistant to targeted BRAF(V600E) inhibition. Our findings suggest that a significant proportion of melanomas arise from nevi. Furthermore, these results demonstrate that PI3K pathway activation serves as a rate-limiting event in this setting, acting at least in part by abrogating OIS. The reactivation of senescence features and elimination of cells refractory to BRAF(V600E) inhibition by PI3K inhibition warrants further investigation into the therapeutic potential of simultaneously targeting these pathways in melanoma.
Pub.: 03 May '12, Pinned: 28 Aug '17
Abstract: BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E). BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors. The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3). The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression. Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment. Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation. In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations. Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD). Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy. For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months. A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab. In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2. Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations. Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.
Pub.: 05 May '12, Pinned: 28 Aug '17
Abstract: The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.
Pub.: 17 Jul '12, Pinned: 28 Aug '17
Abstract: Mutationally activated BRAF(V600E) cooperates with PTEN silencing in the conversion of normal melanocytes to metastatic melanoma cells, but the mechanism underlying this cooperation is poorly understood. Here, the consequences of pharmacologic blockade of BRAF(V600E) or phosphoinositide 3-kinase (PI3K) signaling were explored using pathway-targeted inhibitors and a panel of human BRAF-mutated melanoma-derived cell lines. Blockade of BRAF(V600E) → MEK1/2 → ERK1/2 or class I PI3K inhibited melanoma proliferation, whereas inhibition of AKT had only modest effects, even in cells with mutated or amplified AKT. Although single-agent inhibition of either BRAF(V600E) or PI3K signaling elicited antiproliferative effects, combinatorial inhibition was more potent. Analysis of signaling downstream of BRAF(V600E) or PI3K revealed that these pathways cooperated to regulate protein synthesis through AKT-independent, mTOR complex 1 (mTORC1)-dependent effects on p70(S6K), ribosomal protein S6, and 4E-BP1 phosphorylation. Moreover, inhibition of mTORC1/2 inhibited cell proliferation as profoundly as single-agent inhibition of either BRAF(V600E) or PI3K signaling. These data reveal a mechanism by which BRAF(V600E) and PI3K signaling cooperate to regulate melanoma proliferation through AKT-independent effects on protein translation. Furthermore, this study provides a potential foundation for pathway-targeted combination therapy designed to enhance the therapeutic benefit to patients with melanoma that contain combined alterations in BRAF and PI3K signaling.PI3K, but not AKT, represent potential targets for melanoma therapy.
Pub.: 16 Jan '14, Pinned: 28 Aug '17
Abstract: Macroautophagy (autophagy hereafter) may promote survival and growth of spontaneous tumors, including melanoma. We utilized a genetically engineered mouse model of melanoma driven by oncogenic BrafV600E and deficiency in the Pten tumor suppressor gene in melanocytes to test the functional consequences of loss of the essential autophagy gene autophagy-related-7, Atg7. Atg7 deficiency prevented melanoma development by BrafV600E and allelic Pten loss, indicating that autophagy is essential for melanomagenesis. Moreover, BrafV600E-mutant, Pten-null, Atg7-deficient melanomas displayed accumulation of autophagy substrates and growth defects, which extended animal survival. Atg7-deleted tumors showed increased oxidative stress and senescence, a known barrier to melanomagenesis. Treatment with the BRAF inhibitor dabrafenib decreased tumor growth and induced senescence that was more pronounced in tumors with Atg7 deficiency. Thus, Atg7 promotes melanoma by limiting oxidative stress and overcoming senescence, and autophagy inhibition may be of therapeutic value by augmenting the antitumor activity of BRAF inhibitors.The essential autophagy gene Atg7 promotes development of BrafV600E-mutant, Pten-null melanomas by overcoming senescence, and deleting Atg7 facilitated senescence induction and antitumor activity of BRAF inhibition. This suggests that combinatorial BRAFV600E and autophagy inhibition may improve therapeutic outcomes in patients whose tumors have BRAFV600E/K mutations, an approach currently being explored in clinical trials.
Pub.: 13 Feb '15, Pinned: 28 Aug '17
Abstract: The detection of BRAFV600 mutations in patients with metastatic melanoma is important because of the availability of BRAF inhibitor therapy. However, the clinical relevance of the frequency of BRAFV600 mutant alleles is unclear.Allele frequencies of BRAFV600 mutations were analyzed by ultra-deep next-generation sequencing in formalin-fixed, paraffin-embedded melanoma tissue (75 primary melanomas and 88 matched metastases). In a second study, pretreatment specimens from 76 patients who received BRAF inhibitors were retrospectively analyzed, and BRAFV600 allele frequencies were correlated with therapeutic results.Thirty-five patients had concordantly BRAF-positive and 36 (48%) patients had concordantly BRAF-negative primary melanomas and matched metastases, and four patients had discordant samples with low allele frequencies (3.4-5.2%). Twenty-six of 35 patients with concordant samples had BRAFV600E mutations, three of whom had additional mutations (V600K in two patients and V600R in one) and nine patients had exclusively non-V600E mutations (V600K in eight patients and V600E -c.1799_1800TG > AA- in one patient). The frequency of mutated BRAFV600 alleles was similar in the primary melanoma and matched metastasis in 27/35 patients, but differed by >3-fold in 8/35 of samples. BRAFV600E allele frequencies in pretreatment tumor specimens were not significantly correlated with treatment outcomes in 76 patients with metastatic melanoma who were treated with BRAF inhibitors.BRAFV600 mutation status and allele frequency is consistent in the majority of primary melanomas and matched metastases. A small subgroup of patients has double mutations. BRAFV600 allele frequencies are not correlated with the response to BRAF inhibitors.
Pub.: 27 Oct '15, Pinned: 28 Aug '17
Abstract: The development of resistance to previously effective treatments has been a challenge for health care providers and a fear for patients undergoing cancer therapy. This is an unfortunately frequent occurrence for patients undergoing targeted therapy for tumours harboring the activating V600E mutation of the BRAF gene. Since the initial identification of the BRAF mutation in 2002, a series of small molecular inhibitors that target the BRAFV600E have been developed, but intrinsic and acquired resistance to these drugs has presented an ongoing challenge. More recently, improvements in therapy have been achieved by combining the use of BRAF inhibitors with other drugs, such as inhibitors of the downstream effector mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK). Despite improved success in response rates and in delaying resistance using combination therapy, ultimately, the acquisition of resistance remains a concern. Recent research articles have shed light on some of the underlying mechanisms of this resistance and have proposed numerous strategies that might be employed to overcome or avoid resistance to targeted therapies. This review will explore some of the resistance mechanisms, compare what is known in melanoma cancer to colorectal cancer, and discuss strategies under development to manage the development of resistance.
Pub.: 12 Mar '17, Pinned: 28 Aug '17
Abstract: In the recent past years, many discoveries in the tumor microenvironment have led to changes in the management of melanoma and it is rising up hopes, specially, to those in advanced stages. FDA approved seven new drugs from 2011 to 2014. They are: Vemurafenib, Dabrafenib and Trametinib, kinases inhibitors used for patients that have BRAFV600E mutation; Ipilimumab (anti-CTLA4), Pembrolizumab (anti-PD-1) and Nivolumab (anti-PD-1), monoclonal antibodies that stimulate the immune system; and Peginterferon alfa-2b, an anti-proliferative cytokine used as adjuvant therapy. In this article, we will review the molecular bases for these new metastatic melanoma therapeutic agents cited above and also analyze new molecular discoveries in melanoma study, as Cancer-Testis antigens (CT). They are capable of induce humoral and cellular immune responses in cancer patients and because of this immunogenicity and their restrict expression in normal tissues, they are considered an ideal candidate for vaccine development against cancer. Among CT antigens, NY-ESO-1 is the best characterized in terms of expression patterns and immunogenicity. It is expressed in 20-40% of all melanomas, more in metastatic lesions than in primary ones, and it is very heterogeneous inter and intratumoral. Breslow index is associate with NY-ESO-1 expression in primary cutaneous melanomas, but its relation to patient survival remains controversial.
Pub.: 26 May '17, Pinned: 28 Aug '17
Abstract: A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8-30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.
Pub.: 14 Oct '05, Pinned: 28 Aug '17
Abstract: The rapid emergence of the zebrafish as a cancer model has been aided by advances in genetic, chemical, and imaging technologies. Melanoma in particular highlights both the power and challenges associated with cancer modeling in zebrafish. This chapter focuses on the lessons that have emerged from the melanoma models as paradigmatic of what will apply to nearly all cancer models in the zebrafish system. We specifically focus on methodologies related to germline and mosaic transgenic melanoma generation, and how these can be used to deeply interrogate additional cooperating oncogenes or tumor suppressors. These transgenic tumors can in turn be used to generate zebrafish-specific, stable melanoma cell lines which can be fluorescently labeled, modified by cDNA/CRISPR techniques, and used for detailed in vivo imaging of cancer progression in real time. These zebrafish melanoma models are beginning to elucidate both cell intrinsic and microenvironmental factors in melanoma that have broader implications for human disease. We envision that nearly all of the techniques described here can be applied to other zebrafish cancer models, and likely expanded beyond what we describe here.
Pub.: 18 Jun '16, Pinned: 28 Aug '17
Abstract: Tetraploidy may constitute a metastable state leading to numeric and structural chromosome abnormalities that are associated with cancer. Here, we show that cultured primary p53(-/-) (but not wild type, WT) mouse mammary epithelial cells (MMECs) accumulate a tetraploid sub-population in vitro. This occurs spontaneously, yet can be exacerbated by the addition of microtubule inhibitors as well as of inhibitors of cytokinesis. As compared to WT cells, tetraploid p53(-/-) MMECs contain supernumerary centrosomes and exhibit a reduced propensity to initiate the mitochondrial pathway of apoptosis. Moreover, tetraploid p53(-/-) MMECs are more resistant against anthracyclin-induced cell killing than their diploid counterparts. Altogether, these data indicate that p53 normally suppresses the generation of tetraploid cells, presumably by activating the intrinsic pathway of apoptosis. In the absence of p53, tetraploid cells accumulate as a result of inhibited apoptosis, which contributes to the acquisition of chemotherapy resistance.
Pub.: 04 Apr '09, Pinned: 28 Aug '17
Abstract: In tumorigenesis, aneuploidy is frequently preceded by tetraploidy. Major issues include how tetraploidy arises and how cells can effectively respond to this state. Two recent papers address these issues. Shi and King demonstrate that nondisjunction of chromosomes in mitosis frequently results in tetraploidy through mitotic cleavage failure. Fujiwara et al. demonstrate that p53 null tetraploid cells are highly competent to induce tumors in nude mice. Together, these papers emphasize the unique hazard of tetraploidy and the fact that p53 status has an intrinsic capacity to eliminate tetraploid cells and suppress tumorigenesis. This p53-dependent elimination may represent a checkpoint control.
Pub.: 16 Nov '05, Pinned: 28 Aug '17
Abstract: Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and gamma-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells.
Pub.: 06 May '06, Pinned: 28 Aug '17
Abstract: Aneuploidy is one of the most obvious differences between normal and cancer cells. However, there remains debate over how aneuploid cells arise and whether or not they are a cause or consequence of tumorigenesis. One proposed route to aneuploid cancer cells is through an unstable tetraploid intermediate. Supporting this idea, recent studies demonstrate that tetraploidy promotes chromosomal aberrations and tumorigenesis in vivo. These tetraploid cells can arise by a variety of mechanisms, including mitotic slippage, cytokinesis failure, and viral-induced cell fusion. Furthermore, new studies suggest that there might not be a ploidy-sensing checkpoint that permanently blocks the proliferation of tetraploid cells. Therefore, abnormal division of tetraploid cells might facilitate genetic changes that lead to aneuploid cancers.
Pub.: 28 Feb '07, Pinned: 28 Aug '17
Abstract: Polyploidy, an increased number of chromosome sets, is a surprisingly common phenomenon in nature, particularly in plants and fungi. In humans, polyploidy often occurs in specific tissues as part of terminal differentiation. Changes in ploidy can also result from pathophysiological events that are caused by viral-induced cell fusion or erroneous cell division. Tetraploidization can initiate chromosomal instability (CIN), probably owing to supernumerary centrosomes and the doubled chromosome mass. CIN, in turn, might persist or soon give way to a stably propagating but aneuploid karyotype. Both CIN and stable aneuploidy are commonly observed in cancers. Recently, it has been proposed that an increased number of chromosome sets can promote cell transformation and give rise to an aneuploid tumor. Here, we review how tetraploidy can occur and describe the cellular responses to increased ploidy. Furthermore, we discuss how the specific physiological changes that are triggered by polyploidization might be used as novel targets for cancer therapy.
Pub.: 21 Nov '08, Pinned: 28 Aug '17
Abstract: Tetraploidization has been proposed as an intermediate step toward aneuploidy in human cancer but a general mechanism for the induction of tetraploidy during tumorigenesis is lacking. We report that tetraploidization occurs in p53-deficient cells experiencing a prolonged DNA damage signal due to persistent telomere dysfunction. Live-cell imaging revealed that these cells have an extended G2 due to ATM/ATR- and Chk1/Chk2-mediated inhibition of Cdk1/CyclinB and eventually bypass mitosis. Despite their lack of mitosis, the cells showed APC/Cdh1-dependent degradation of the replication inhibitor geminin, followed by accumulation of Cdt1, which is required for origin licensing. Cells then entered a second S phase resulting in whole-genome reduplication and tetraploidy. Upon restoration of telomere protection, these tetraploid cells resumed cell division cycles and proliferated. These observations suggest a general mechanism for the induction of tetraploidization in the early stages of tumorigenesis when telomere dysfunction can result from excessive telomere shortening.
Pub.: 08 Apr '10, Pinned: 28 Aug '17
Abstract: Loss of RASSF1A leads to several mitotic abnormalities, including cytokinesis failure and tetraploidization. Uncontrolled proliferation of tetraploid cells is known to trigger genomic instability and tumor development and is normally prevented through activation of a p53-dependent tetraploidy checkpoint. RASSF1A is the most commonly silenced and p53 is the most frequently mutated tumor suppressor gene in human cancer. However, their mutual contribution to tumorigenesis has never been investigated in animal models. Here, we explore whether concomitant loss of RASSF1A and p53 will result in increased levels of aneuploidy, genomic instability and tumorigenesis. We have intercrossed Rassf1a-knockout mice with mice lacking the p53 gene and generated a combination of single- and compound-mutant animals. Rassf1a(-/-) p53(-/-) mice were viable and fertile and developed normally. However, these mice were remarkably tumor prone and succumbed to malignancies significantly faster than single-mutant littermates, with a median survival time of 136 days (versus 158 days in p53(-/-) mice, P=0.0207, and >600 days in Rassf1a(-/-) animals, P<0.0001). Rassf1a-null mice with one functional p53 allele displayed a more moderate, yet tumor-prone phenotype, characterized by increased tumor multiplicity as compared with single knockouts. On cell-cycle profiling and cytogenetic analysis, cells derived from Rassf1a(-/-) p53(-/-) mice exhibited several mitotic defects associated with high levels of tetraploidy/aneuploidy. Conversely, cells with a proficient p53 allele could better cope with the mitotic failures imposed by Rassf1a loss. Altogether, we provide the first experimental evidence for a pivotal role of Rassf1a as an early 'gatekeeper' gene, whose loss of function deteriorates cellular fitness by enhancing tetraploidization. Concomitant loss of p53, which causes unrestrained propagation of tetraploids into aneuploid cells, further undermines genomic stability and accelerates tumorigenesis.
Pub.: 05 Oct '10, Pinned: 28 Aug '17
Abstract: Aneuploidy, often preceded by tetraploidy, is one of the hallmarks of solid tumors. Indeed, both aneuploidy and tetraploidy are oncogenic occurrences that are sufficient to drive neoplastic transformation and cancer progression. True to form, the tumor suppressor p53 obstructs propagation of these dangerous chromosomal events by either instigating irreversible cell cycle arrest or apoptosis. The tumor suppressor Lats2, along with other tumor inhibitory proteins such as BRCA1/2 and BubR1, are central to p53-dependent elimination of tetraploid cells. Not surprisingly, these proteins are frequently inactivated or downregulated in tumors, synergizing with p53 inactivation to establish an atmosphere of "tolerance" for a non-diploid state.
Pub.: 20 Aug '11, Pinned: 28 Aug '17
Abstract: Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy.
Pub.: 10 May '12, Pinned: 28 Aug '17
Abstract: Data from human tumors and mouse models suggest that tetraploidy, one example of polyploidy, can promote tumorigenesis. In this issue of Cancer Cell, Davoli and De Lange make important connections between tetraploidy, tumorigenesis, and telomere crisis-a common event during the development of human cancers.
Pub.: 16 Jun '12, Pinned: 28 Aug '17
Abstract: Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the "tetraploidy checkpoint", p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin).
Pub.: 08 Nov '14, Pinned: 28 Aug '17
Abstract: To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.
Pub.: 29 Apr '15, Pinned: 28 Aug '17
Abstract: Several lines of evidence indicate that whole-genome duplication resulting in tetraploidy facilitates carcinogenesis by providing an intermediate and metastable state more prone to generate oncogenic aneuploidy. Here, we report a novel strategy to preferentially kill tetraploid cells based on the abrogation of the spindle assembly checkpoint (SAC) via the targeting of TTK protein kinase (better known as monopolar spindle 1, MPS1). The pharmacological inhibition as well as the knockdown of MPS1 kills more efficiently tetraploid cells than their diploid counterparts. By using time-lapse videomicroscopy, we show that tetraploid cells do not survive the aborted mitosis due to SAC abrogation upon MPS1 depletion. On the contrary diploid cells are able to survive up to at least two more cell cycles upon the same treatment. This effect might reflect the enhanced difficulty of cells with whole-genome doubling to tolerate a further increase in ploidy and/or an elevated level of chromosome instability in the absence of SAC functions. We further show that MPS1-inhibited tetraploid cells promote mitotic catastrophe executed by the intrinsic pathway of apoptosis, as indicated by the loss of mitochondrial potential, the release of the pro-apoptotic cytochrome c from mitochondria, and the activation of caspases. Altogether, our results suggest that MPS1 inhibition could be used as a therapeutic strategy for targeting tetraploid cancer cells.
Pub.: 08 Dec '15, Pinned: 28 Aug '17
Abstract: The genes required for stem cell specification and lineage restriction during embryogenesis also play fundamental roles in adult tissue regeneration and cancer. This "development-regeneration-cancer" axis is exemplified by the vertebrate pigmentation system. Melanocytes exhibit almost unlimited self-renewal capacity during regenerative processes such as mammalian hair recoloration and zebrafish fin regeneration. Melanoma utilizes many regulatory signals and pathways required during ontogeny and regeneration. A discussion of these interconnections highlights how studies of stem cell function in embryonic and regenerative contexts can yield insights into melanoma biology.
Pub.: 13 Sep '08, Pinned: 28 Aug '17
Abstract: Advantageous organismal and technical attributes of the zebrafish are being increasingly applied to study cancer biology. Along with other tumor models, zebrafish that develop melanomas have been generated. In both genetics and phenotype, zebrafish melanomas are strikingly similar to their human counterparts. For this reason, studies in the zebrafish are poised to make significant contributions to melanoma biology. In this review, we summarize important features of human melanoma and discuss how the zebrafish can be used to address many questions that remain unanswered about this devastating disease.
Pub.: 13 Jan '09, Pinned: 28 Aug '17
Abstract: Deregulated Ras signalling is implicated in most human neoplasia, exemplified by melanoma. Whereas Raf activation occurs almost ubiquitously in benign and malignant melanocytic neoplasms, implying an involvement in tumour initiation, phosphoinositide 3-kinase (PI3K) activation occurs predominantly in malignant neoplasms, implying an involvement in malignant progression. Here, we dissect the contributions of these two pathways to tumourigenesis in vivo, by modulating their activities in zebrafish melanocytes. Misexpression of oncogenic Ras (V12RAS) in founder fish induced frequent melanoma, beginning at larval stages, with concomitant activation of Raf-Mek-Erk and PI3K-Akt signalling. Misexpression of effector-domain mutants of V12RAS, or of various downstream effectors, confirmed a selective role for the Raf-Mek-Erk pathway in initiating neoplasia, but highlighted the requirement for additional Ras effector pathways for malignancy. The phenotype of animals with germ-line transmission of V12RAS resembled familial atypical mole and melanoma (FAMM) syndrome: melanocytes displayed hyperplasia, dysplasia, altered terminal differentiation and spontaneously progressed to invasive melanoma. Co-expressing a dominant-interfering form of PI3K abolished V12RAS-induced malignancy, demonstrating a direct role for PI3K signalling in the malignant progression of melanoma in vivo, and highlighting PI3K as a promising target for melanoma therapy.
Pub.: 28 May '09, Pinned: 28 Aug '17
Abstract: The zebrafish has emerged as a powerful model system to study human diseases, including a variety of neoplasms. Principal components that have contributed to the rise in use of this vertebrate model system are its high fecundity, ease of genetic manipulation, and low cost of maintenance. Vital imaging of the zebrafish is possible from the transparent embryonic stage through adulthood, the latter enabled by a number of mutant lines that ablate pigmentation. As a result, high-resolution analyses of tumor progression can be accomplished in vivo. Straightforward transgenesis of zebrafish has been employed to develop numerous tumor models that recapitulate many aspects of human neoplastic disease, both in terms of pathologic and molecular conservation. The small size of zebrafish embryos has enabled screens for novel chemotherapeutic agents. Its facile genetics have been exploited in studies that extend beyond modeling cancer to investigations that define new cancer genes and mechanisms of cancer progression. Together, these attributes have established the zebrafish as a robust and versatile model system for investigating cancer. In this chapter we describe methods that are used to study a gene's impact on melanoma progression. We detail methods for making transgenic animals and screening for tumor onset as well as methods to investigate tumor invasion and propagation.
Pub.: 18 Jul '14, Pinned: 28 Aug '17
Abstract: Melanocyte development provides an excellent model for studying more complex developmental processes. Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body. The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development. In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches. This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma.
Pub.: 12 Feb '15, Pinned: 28 Aug '17
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