Aneuploidy and Tetraploidy as Distinct Patterns During Melanomagenesis.

Melanoma is characterized by a high degree of chromosome instability (CIN), the loss or gain of entire chromosomes or pieces of chromosomes. Also, CIN is likely to drive the progression of benign melanocytic lesions to malignant tumors, although very little is known about the acquisition of the mechanisms that promote CIN along this progression. Here, we describe the development of a model system to study the progression of melanomagenesis starting with normal human melanocytes followed by inactivation of the p53 and pRb tumor suppressors by addition of the E6/E7 proteins. The cells were then transduced with a growth-promoting, constitutionally-active mutant NRAS. The addition of E6/E7 and E6/E7 NRAS was found to give a growth advantage to the cells compared to normal melanocytes and a statistically significant gain of aneuploidy; aneuploidy was 24.7% in primary melanocytes, 33.8% in E6/E7 melanocytes, and 70.5% in E6/E7 NRAS melanocytes. Further, we found an increase in tetraploid cells in the cell model which was statistically significant between primary melanocytes and E6/E7, NRAS melanocytes. We also observed an increase in aneuploid cells between three population doublings in primary melanocytes, whereas this increase was not seen in the E6/E7 melanocytes. Together, these data demonstrate that this model system utilizing stepwise addition of genetic mutations driving melanomagenesis is a useful tool to study CIN and could even be used to study the mechanisms responsible for these alterations in genetic makeup.

How, and from which cell sources, do nevi really develop?

Melanocytic neoplasms are a diverse group of benign and malignant tumors with variable clinical features. While some models still promote the epidermal melanocyte as the origin of melanocytic neoplasms, clinical findings are inconsistent with this theory for the majority of tumors. Despite advances in naevus and melanoma biology, the location and differentiation status of the cell of origin remains undefined. Germ line genetics, biological state and cellular location of the mutated cell, as well as local environmental factors all likely play a role in the development of melanocytic neoplasms. Herein, we will review potential models for melanocytic neoplasia and discuss research challenges and opportunities.

Potential role of meiosis proteins in melanoma chromosomal instability.

Melanomas demonstrate chromosomal instability (CIN). In fact, CIN can be used to differentiate melanoma from benign nevi. The exact molecular mechanisms that drive CIN in melanoma have yet to be fully elucidated. Cancer/testis antigens are a unique group of germ cell proteins that are found to be primarily expressed in melanoma as compared to benign nevi. The abnormal expression of these germ cell proteins, normally expected only in the testis and ovaries, in somatic cells may lead to interference with normal cellular pathways. Germ cell proteins that may be particularly critical in CIN are meiosis proteins. Here, we review pathways unique to meiosis with a focus on how the aberrant expression of meiosis proteins in normal mitotic cells "meiomitosis" could impact chromosomal instability in melanoma and other cancers.

Selective inhibition of p300 HAT blocks cell cycle progression, induces cellular senescence, and inhibits the DNA damage response in melanoma cells.

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to have critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

Germ cell proteins in melanoma: prognosis, diagnosis, treatment, and theories on expression.

Germ cell protein expression in melanoma has been shown to correlate with malignancy, severity of disease and to serve as an immunologic target for therapy. However, very little is known about the role that germ cell proteins play in cancer development. Unique germ cell pathways include those involved in immortalization, genetic evolution, and energy metabolism. There is an ever increasing recognition that within tumors there is a subpopulation of cells with stem-cell-like characteristics that play a role in driving tumorgenesis. Stem cell and germ cell biology is intertwined. Given the enormous potential and known expression of germ cell proteins in melanoma, it is possible that they represent a largely untapped resource that may play a fundamental role in tumor development and progression. The purpose of this paper is to provide an update on the current value of germ cell protein expression in melanoma diagnosis, prognosis, and therapy, as well as to review critical germ cell pathways and discuss the potential roles these pathways may play in malignant transformation.

Diagnostic role of chromosomal instability in melanoma.

Early diagnosis gives melanoma patients the best chance for long term survival. However discrimination of an early melanoma from an unusual/atypical benign nevus can represent a significant challenge. There are no current pathological markers to definitively define malignant potential in these indeterminate lesions. Thus, there is a need for improved diagnostic tools. Chromosomal instability (CIN) is a hallmark of cancer and is markedly prevalent in melanoma. Advances in genomics have opened the door for the development of molecular tools to better segregate benign and malignant lesions. This paper focuses on CIN in melanoma and the role of current diagnostic approaches.

Neuropilin-2: a novel biomarker for malignant melanoma?

Neuropilin-2, a cell surface receptor involved in angiogenesis and axonal guidance, has recently been shown to be a critical mediator of tumor-associated lymphangiogenesis. Given that lymphangiogenesis is a major conduit of metastasis in melanomas and that blocking neuropilin-2 function in vivo is effective in inhibiting tumor cell metastasis, we sought to determine the clinical relevance of neuropilin-2 expression in cutaneous melanoma. Immunohistochemical analysis of neuropilin-2 expression was evaluated in nevomelanocytic proliferations that included a tissue microarray and histologic sections from samples of primary melanomas (n = 42; 40 for tissue microarray, 2 for histologic sections), metastatic melanomas (n = 30; 22 for tissue microarray, 8 for histologic sections), and nevi (n = 30; 5 for tissue microarray, 25 for histologic sections), as well as a panel of normal human tissues and select nonmelanocytic tumors. Staining for grading and intensity of neuropilin-2 expression was estimated semiquantitatively as follows for the former: less than 20%, 20% to 60%, and more than 60% of tissue present, and for the latter from 0 to 3, with 3 being the highest and 0 the lowest intensity. In nevomelanocytic proliferations, more than 20% staining for neuropilin-2 was noted in 36 (86%) of 42 cases of primary melanoma, in 27 (90%) of 30 cases of metastatic melanoma, and in 9 (30%) of 30 cases of nevi with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (P < .0001). For staining intensity, an intensity of 2 or more was noted in 36 (86%) of 42 cases of primary melanoma, in 17 (57%) of 30 cases of metastatic melanoma and in 7 (30%) of 23 cases of nevi, with differences achieving statistical significance between melanoma (primary and metastatic) and nevi (P < .0001). In normal human tissue, consistently strong neuropilin-2 staining was noted in kidney (glomerular endothelial cells, collecting tubules, and collecting ducts), skin (epidermal keratinocytes), and testes (epithelium of the seminiferous tubules), whereas in tumoral tissue, consistently strong staining was noted only in renal cell carcinoma but not in any of the other tumors studied. More recently, using a heterotypic coculture methodology with melanoma and endothelial cells, we have demonstrated successful up-regulation of neuropilin-2 and confirmed the critical role of neuropilin-2 in melanoma-endothelial interactions. Because these coculture methods were developed to model melanoma metastasis, the significantly increased and enhanced expression of neuropilin-2 staining in primary and metastatic melanoma versus nevi in the current study suggests that it is also relevant in vivo.

Radiosensitization of mammary carcinoma cells by telomere homolog oligonucleotide pretreatment.

INTRODUCTION: Ionizing radiation (IR) is a widely used approach to cancer therapy, ranking second only to surgery in rate of utilization. Responses of cancer patients to radiotherapy depend in part on the intrinsic radiosensitivity of the tumor cells. Thus, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. METHODS: Mammary tumor cells were treated by a 16-base phosphodiester-linked oligonucleotide homologous to the telomere G-rich sequence TTAGGG (T-oligo: GGTTAGGTGTAGGTTT) or a control-oligo (the partial complement, TAACCCTAACCCTAAC) followed by IR. The inhibition of tumor cell growth in vitro was assessed by cell counting and clonogenic cell survival assay. The tumorigenesis of tumor cells after various treatments was measured by tumor growth in mice. The mechanism underlying the radiosensitization by T-oligo was explored by immunouorescent determination of phosphorylated histone H2AX (γH2AX) foci, ß-galactosidase staining, comet and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assays. The efficacy of the combined treatment was assessed in a spontaneous murine mammary tumor model. RESULTS: Pretreatment of tumor cells with T-oligo for 24 hours in vitro enhanced both senescence and apoptosis of irradiated tumor cells and reduced clonogenic potential. Radiosensitization by T-oligo was associated with increased formation and/or delayed resolution of γH2AX DNA damage foci and fragmented DNA. T-oligo also caused radiosensitization in two in vivo mammary tumor models. Indeed, combined T-oligo and IR-treatment in vivo led to a substantial reduction in tumor growth. Of further significance, treatment with T-oligo and IR led to synergistic inhibition of the growth of spontaneous mammary carcinomas. Despite these profound antitumor properties, T-oligo and IR caused no detectable side effects under our experimental conditions. CONCLUSIONS: Pretreatment with T-oligo sensitizes mammary tumor cells to radiation in both in vitro and in vivo settings with minimal or no normal tissue side effects.

Telomeric DNA induces p53-dependent reactive oxygen species and protects against oxidative damage.

BACKGROUND: Reactive oxygen species (ROS) are generated by cellular metabolism as well as by exogenous agents. While ROS can promote cellular senescence, they can also act as signaling molecules for processes that do not lead to senescence. Telomere homolog oligonucleotides (T-oligos) induce adaptive DNA damage responses including increased DNA repair capacity and these effects are mediated, at least in part, through p53. OBJECTIVE: Studies were undertaken to determine whether such p53-mediated protective responses include enhanced antioxidant defenses. METHODS: Normal human fibroblasts as well as R2F fibroblasts expressing wild type or dominant negative p53 were treated with an 11-base T-oligo, a complementary control oligo or diluents alone and then examined by western blot analysis, immunofluorescence microscopy and various biochemical assays. RESULTS: We now report that T-oligo increases the level of the antioxidant enzymes superoxide dismutase 1 and 2 and protects cells from oxidative damage; and that telomere-based gammaH2AX (DNA damage) foci that form in response to T-oligos contain phosphorylated ATM and Chk2, proteins known to activate p53 and to mediate cell cycle arrest in response to oxidative stress. Further, T-oligo increases cellular ROS levels via a p53-dependent pathway, and these increases are abrogated by the NAD(P)H oxidase inhibitor diphenyliodonium chloride. CONCLUSION: These results suggest the existence of innate telomere-based protective responses that act to reduce oxidative damage to cells. T-oligo treatment induces the same responses and offers a new model for studying intracellular ROS signaling and the relationships between DNA damage, ROS, oxidative stress, and cellular defense mechanisms.

Telomere-mediated effects on melanogenesis and skin aging.

UV-induced melanogenesis (tanning) and "premature aging" or photoaging result in large part from DNA damage. This article reviews data tying both phenomena to telomere-based DNA damage signaling and develops a conceptual framework in which both responses may be understood as cancer-avoidance protective mechanisms.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 25-31; doi:10.1038/jidsymp.2009.9.

Telomere homolog oligonucleotides induce apoptosis in malignant but not in normal lymphoid cells: mechanism and therapeutic potential.

Human B- or T-cell lymphoma lines and primary murine lymphomas were treated with DNA oligonucleotides homologous to the telomere (TTAGGG repeat; "T-oligo"), either alone or in combination with standard, widely-used anticancer chemotherapeutic agents. T-oligo induces cell cycle arrest and apoptosis in cultured human or murine B or T-lymphoma cell lines and primary tumor cells, but exerts no detectable toxicity on normal human or murine primary lymphocytes. Exposure to T-oligo is hypothesized to mimic exposure of the 3' telomere repeat sequence, activating the ataxia telangiectasia mutated kinase, which phosphorylates downstream effectors such as p53, but effects are not dependent solely on functional p53. T-oligo causes early S-phase arrest and cooperates well with G(2)- or M-phase-specific anticancer agents; when combined at 1/10th of the conventional dose, vincristine and T-oligo produce greater-than-additive killing of human or murine lymphoma cells (78% of cells undergoing apoptosis after 6 hr vs. 5% of control cells). In mice, 1/10th of the conventional dose of a standard combination of cyclophosphamide, adriamycin, vincristine and prednisone is twice as effective when used in combination with low dose T-oligo. Thus, T-oligo sensitizes tumors to traditional anticancer agents and represents a potentially important new addition to the therapeutic arsenal for aggressive lymphomas.

Photoprotection in human skin--a multifaceted SOS response.

Human skin has developed elaborate defense mechanisms for combating a wide variety of potentially damaging environmental factors; principal among these is UV light. Despite these defenses, short-term damage may include painful sunburn and long-term UV damage results in both accelerated skin aging and skin cancers such as basal cell carcinoma, squamous cell carcinoma and even malignant melanoma. While UV radiation damages many cellular constituents, its most lasting effects involve DNA alteration. The following sections briefly review UV-inducible protective responses in bacteria and in skin, thymidine dinucleotides (pTT) as a powerful probe of DNA damage responses, and potential means of harnessing these inducible responses therapeutically to reduce the now enormous burden of cutaneous photodamage in our society.

Oligonucleotide treatment increases eumelanogenesis, hair pigmentation and melanocortin-1 receptor expression in the hair follicle.

It was previously reported that telomere homologue oligonucleotides (T-oligos) can induce a variety of cellular responses in skin including increased melanogenesis. To assess the effects of T-oligos on hair pigmentation, we administered thymidine dinucleotide (pTT), one-third of the TTAGGG telomere repeat sequence, intradermally at distinct time points of the depilation-induced hair cycle in C3H/HeJ mice. Penetration of T-oligos into the hair follicle (HF) was monitored by using FITC-labelled pTT and confocal microscopy. pTT treatment on days 1-5 after depilation, during early anagen, did not significantly alter the number and proliferation of melanocytes (Trp-2-positive cells), compared with vehicle-treated controls. However, pTT treatment on days 5-12 after depilation, during mid- to late anagen, resulted in the formation of darker hairs, that showed a significantly increased eumelanin/total melanin ratio in their sub-apical agouti band region, compared with vehicle-treated controls (P < 0.05). By RT-PCR and western blot, full thickness skin of pTT-treated mice showed increases in Trp-1, Trp-2 and tyrosinase mRNA and protein levels, compared with control mice. Western blot analyses of two receptors that positively regulate eumelanogenesis, melanocortin type 1 receptor (MC-1R) and kit, showed increased expression of MC-1R protein in pTT-treated versus control skin, while the levels of c-kit receptor remained unchanged. These data demonstrate that pTT treatment increases eumelanogenesis in HFs, associated with increased tyrosinase, TRP-1 and MC-1R expression. These data also raise the possibility of using T-oligos to modulate hair pigmentation.

Telomere 3' overhang-specific DNA oligonucleotides induce autophagy in malignant glioma cells.

Telomere 3' overhang-specific DNA oligonucleotides (T-oligos) induce cell death in cancer cells, presumably by mimicking telomere loop disruption. Therefore, T-oligos are considered an exciting new therapeutic strategy. The purpose of this study was to elucidate how T-oligos exert antitumor effects on human malignant glioma cells in vitro and in vivo. We demonstrated that T-oligos inhibited the proliferation of malignant glioma cells through induction of nonapoptotic cell death and mitochondria hyperpolarization, whereas normal astrocytes were resistant to T-oligos. Tumor cells treated with T-oligos developed features compatible with autophagy, with development of autophagic vacuoles and conversion of an autophagy-related protein, microtubule-associated protein 1 light chain 3 from type I (cytoplasmic form) to type II (membrane form of autophagic vacuoles). A reverse-phase protein microarray analysis and Western blotting revealed that treatment with T-oligos inhibited the mammalian target of the rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment with T-oligos significantly prolonged the survival time of mice inoculated intracranially with malignant glioma cells compared with that of untreated mice and those treated with control oligonucleotides (P=0.0065 and P=0.043, respectively). These results indicate that T-oligos stimulate the induction of nonapoptotic autophagic also known as type II programmed cell death and are thus promising in the treatment of malignant glioma.

Telomeric DNA induces apoptosis and senescence of human breast carcinoma cells.

INTRODUCTION: Cancer is a leading cause of death in Americans. We have identified an inducible cancer avoidance mechanism in cells that reduces mutation rate, reduces and delays carcinogenesis after carcinogen exposure, and induces apoptosis and/or senescence of already transformed cells by simultaneously activating multiple overlapping and redundant DNA damage response pathways. METHODS: The human breast carcinoma cell line MCF-7, the adriamycin-resistant MCF-7 (Adr/MCF-7) cell line, as well as normal human mammary epithelial (NME) cells were treated with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos). SCID mice received intravenous injections of MCF-7 cells followed by intravenous administration of T-oligos. RESULTS: Acting through ataxia telangiectasia mutated (ATM) and its downstream effectors, T-oligos induced apoptosis and senescence of MCF-7 cells but not NME cells, in which these signaling pathways were induced to a far lesser extent. In MCF-7 cells, experimental telomere loop disruption caused identical responses, consistent with the hypothesis that T-oligos act by mimicking telomere overhang exposure. In vivo, T-oligos greatly prolonged survival of SCID mice following intravenous injection of human breast carcinoma cells. CONCLUSION: By inducing DNA damage-like responses in MCF-7 cells, T-oligos provide insight into innate cancer avoidance mechanisms and may offer a novel approach to treatment of breast cancer and other malignancies.

Features that determine telomere homolog oligonucleotide-induced therapeutic DNA damage-like responses in cancer cells.

Cancer is the second leading cause of death in the USA, with metastatic disease proving a particular management challenge. Treatment modalities for patients with metastatic disease are limited, and survival beyond 5 years is uncommon. We have reported that an 11-base DNA oligonucleotide 100% homologous to the telomere 3' overhang can induce apoptosis, senescence and/or differentiation of several types of malignant cells in vitro and in vivo, while having minimal effect on normal cells. We now report that 22 oligonucleotides, 9-20 bases in length, with or without a 5' phosphate group and with varying homology (40-100%) to the 3' overhang, inhibit growth and induce apoptosis of human cell lines derived from breast cancers, pancreatic and ovarian carcinomas, and malignant melanoma, lines that lack p53 and/or p16 and harbor a variety of other abnormalities in key regulatory signaling pathways. Cytosine (C) content adversely affected oligonucleotide efficacy, decreasing their effect on cellular apoptosis by > or =80%. These data confirm and expand our earlier work suggesting that such telomere homolog oligonucleotides (T-oligos) target an innate anti-cancer defense system in human cells and may provide an effective treatment for cancers of multiple different cellular origins and genetic profile.

A role for WRN in telomere-based DNA damage responses.

Telomeres cap the ends of eukaryotic chromosomes and prevent them from being recognized as DNA breaks. We have shown that certain DNA damage responses induced during senescence and, at times of telomere uncapping, also can be induced by treatment of cells with small DNA oligonucleotides homologous to the telomere 3' single-strand overhang (T-oligos), implicating this overhang in generation of these telomere-based damage responses. Here, we show that T-oligo-treated fibroblasts contain gammaH2AX foci and that these foci colocalize with telomeres. T-oligos with nuclease-resistant 3' ends are inactive, suggesting that a nuclease initiates T-oligo responses. We therefore examined WRN, a 3'-->5' exonuclease and helicase mutated in Werner syndrome, a disorder characterized by aberrant telomere maintenance, premature aging, chromosomal rearrangements, and predisposition to malignancy. Normal fibroblasts and U20S osteosarcoma cells rendered deficient in WRN showed reduced phosphorylation of p53 and histone H2AX in response to T-oligo treatment. Together, these data demonstrate a role for WRN in processing of telomeric DNA and subsequent activation of DNA damage responses. The T-oligo model helps define the role of WRN in telomere maintenance and initiation of DNA damage responses after telomere disruption.