Promotes Melanoma Cell Growth Research Articles

DDB2 promotes melanoma cell growth by transcriptionally regulating the expression of KMT2A and predicts a poor prognosis.

Identification of potential key targets of melanoma, a fatal skin malignancy, is critical to the development of new cancer therapies. Lysine methyltransferase 2A (KMT2A) promotes melanoma growth by activating the human telomerase reverse transcriptase (hTERT) signaling pathway; however, the exact mechanism remains elusive. This study aimed to reveal new molecular targets that regulate KMT2A expression and melanoma growth. Using biotin-streptavidin-agarose pull-down and proteomics, we identified Damage-specific DNA-binding protein 2 (DDB2) as a KMT2A promoter-binding protein in melanoma cells and validated its role as a regulator of KMT2A/hTERT signaling. DDB2 knockdown inhibited the expression of KMT2A and hTERT and inhibited the growth of melanoma cells invitro. Conversely, overexpression of DDB2 activated the expression of KMT2A and promoted the growth of melanoma cells. Additionally, we demonstrated that DDB2 expression was higher in tumor tissues of patients with melanoma than in corresponding normal tissues and was positively correlated with KMT2A expression. Kaplan-Meier analysis showed a poor prognosis in patients with high levels of DDB2 and KMT2A. Overall, our data suggest that DDB2 promotes melanoma cell growth through the transcriptional regulation of KMT2A expression and predicts poor prognosis. Therefore, targeting DDB2 may regulate the effects of KMT2A on melanoma growth and progression, providing a new potential therapeutic strategy for melanoma.

Arginyl-tRNA-protein transferase 1 (ATE1) promotes melanoma cell growth and migration.

Arginyl-tRNA-protein transferase 1 (ATE1) catalyses N-terminal protein arginylation, a post-translational modification implicated in cell migration, invasion and the cellular stress response. Herein, we report that ATE1 is overexpressed in NRAS-mutant melanomas, while it is downregulated in BRAF-mutant melanomas. ATE1 expression was higher in metastatic tumours, compared with primary tumours. Consistent with these findings, ATE1 depletion reduced melanoma cell viability, migration and colony formation. Reduced ATE1 expression also affected cell responses to mTOR and MEK inhibitors and to serum deprivation. Among putative ATE1 substrates is the tumour suppressor AXIN1, pointing to the possibility that ATE1 may fine-tune AXIN1 function in melanoma. Our findings highlight an unexpected role for ATE1 in melanoma cell aggressiveness and suggest that ATE1 constitutes a potential new therapeutic target.

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

UBTF facilitates melanoma progression via modulating MEK1/2-ERK1/2 signalling pathways by promoting GIT1 transcription

BackgroundUBTF is an HMGB-box DNA binding protein and a necessary Pol I/Pol II basal transcription factor. It has been found that UBTF involves in carcinogenesis and progression of a few cancers. Nevertheless, the the biological function and potential molecular mechanism of UBTF in melanoma are still not clear and need to be clarified.MethodsUBTF and GIT1 expressions in melanoma specimens and cell lines were examined by quantitative real-time PCR (qRT-PCR) and Western blot. MTT and colony formation assays were used to investigate the effects of UBTF and GIT1 on melanoma cell proliferation. Cell cycle and apoptosis assays were detected by flow cytometry. Tumor formation assay was used to analyze the effect of UBTF on melanoma growth. Bioinformatics predicting, chromatin immunoprecipitation (ChIP)-qRT-PCR and reporter gene assay were fulfilled for verifing GIT1 as UBTF targeting gene.ResultsHere we reported that UBTF mRNA and protein expressions were upregulated in primary melanoma specimens and cell lines. UBTF overexpression facilitated melanoma cell proliferation and cell cycle progression and restrained. Silencing UBTF suppressed cell multiplication, cell cycle progression and tumor growth, and promoted apoptosis. UBTF expression was positively related with GIT1 expression in human melanoma tissues. It was verified that UBTF promoted GIT1 transcription in melanoma cells through binding to the promoter region of GIT1. Furthermore, GIT1 overexpression promoted melanoma cell growth and suppressed apoptosis. Knockdown of GIT1 inhibited cell multiplication and induced apoptosis. Overexpression of GIT1 eliminated the effects of silencing UBTF on melanoma cells. Importantly, UBTF activated MEK1/2-ERK1/2 signalling pathways by upregulating GIT1 expression.ConclusionsOur study demonstrates that UBTF promotes melanoma cell proliferation and cell cycle progression by promoting GIT1 transcription, thereby activating MEK1/2-ERK1/2 signalling pathways. The findings indicate that UBTF plays a crucial function in melanoma and may be a potential therapeutic target for the treatment of this disease.

CD147 promotes melanoma cell growth via SOX4-mediated glycolytic metabolism

Purpose: To determine the functional roles of cluster of differentiation 147 (CD147) in glycolysis in melanoma cells.Methods: CD147 expression in melanoma tissue and adjacent normal tissue was determined using quantitative real time polymrase chain reaction (qRT-PCR) and immunohistochemistry. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate cell viability and colony formation, respectively. The role of CD147 in glycolysis in melanoma cells was investigated by determining glucose uptake, production of lactate, and cellular level of ATP.Results: CD147 was enhanced more in melanoma tissue than that in the adjacent normal tissue (p < 0.001). CD147 overexpression promoted the viability and colony formation of melanoma cells. On the other hand, CD147 silencing decreased the viability and colony formation of melanoma cells. Glucose uptake, production of lactate, and cellular level of ATP were upregulated in melanoma cells by CD147 overexpression and downregulated by shRNA-mediated depletion of CD147. CD147 increased expression of C-X-C motif chemokine ligand 1 (CXCL1) to activate the sex-determining region Y-related high-mobility group box 4 (SOX4) pathway. Knockdown of CXCL1 attenuated the positive regulatory effect of CD147 on SOX4. Besides, overexpression of SOX4 reversed the suppressive effects of CD147 silencing on melanoma cell viability, colony formation, and glycolysis.Conclusion: CD147 contributes to melanoma cell growth via upregulation of SOX-mediated glycolysis, thus providing a therapeutic avenue for the management of melanoma.
 Keywords: Cluster of differentiation 147, CD147, Sex-determining region Y-related high-mobility group box 4, Melanoma, Cell growth, Glycolysis

Long non-coding RNA LINC00665 promotes melanoma cell growth and migration via regulating the miR-224-5p/VMA21 axis.

Melanoma is an aggressive malignant skin tumor endangering the health of patients. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been increasingly reported to be implicated in the carcinogenesis of melanoma. Long intergenic non-coding RNA 00665 (LINC00665) has been found to exert important regulatory roles in some cancers, yet its function in melanoma remains to be investigated. QRT-PCR analysis was conducted to evaluate the relative expression of RNAs. Functional experiments in vitro including colony formation, EdU, wound-healing and transwell assays, as well as in vivo xenograft assays, were utilized to study the role of LINC00665 in melanoma. Mechanical experiments were implemented to probe into the molecular linkage of LINC00665, miR-224-5p and VMA21. LINC00665 was abnormally highly expressed in melanoma cells. Silencing LINC00665 could inhibit the proliferation and migration of melanoma cells. LINC00665 sponged miR-224-5p to upregulate VMA21. VMA21 knockdown exerted similarly interfering effects on above biological processes in melanoma cells. However, VMA21 overexpression abolished the in vitro and in vivo outcomes of LINC00665 silencing. LINC00665 promotes proliferative and migrating abilities of melanoma cells via targeting miR-224-5p/VMA21 axis.

Interplay between small and long non-coding RNAs in cutaneous melanoma: a complex jigsaw puzzle with missing pieces.

The incidence of cutaneous melanoma (CM) has increased in the past few decades. The biology of melanoma is characterized by a complex interaction between genetic, environmental and phenotypic factors. A greater understanding of the molecular mechanisms that promote melanoma cell growth and dissemination is crucial to improve diagnosis, prognostication, and treatment of CM. Both small and long non‐coding RNAs (lncRNAs) have been identified to play a role in melanoma biology; microRNA and lncRNA expression is altered in transformed melanocytes and this in turn has functional effects on cell proliferation, apoptosis, invasion, metastasis, and immune response. Moreover, specific dysregulated ncRNAs were shown to have a diagnostic or prognostic role in melanoma and to drive the establishment of drug resistance. Here, we review the current literature on small and lncRNAs with a role in melanoma, with the aim of putting into some order this complex jigsaw puzzle.

BANCR contributes to the growth and invasion of melanoma by functioning as a competing endogenous RNA to upregulate Notch2 expression by sponging miR‑204.

BRAF-activated non-coding RNA (BANCR) is a long non-coding RNA (lncRNA) that contributes to the initiation and development of many solid tumors, including melanoma. However, the BANCR functions and downstream mechanisms are largely unknown. In this study, we aim to investigate how BANCR participates in the proliferation and migration of malignant melanoma and elucidate the underlying mechanism in this process. We found that the expression of the BANCR was low in melanocytic nevus and human melanocytes but high in melanoma tissues and cell lines. Knockdown of BANCR inhibited melanoma cell proliferation and invasion, and induced cell apoptosis. The decreased expression of relative marker proteins further demonstrated the inhibitory effect of BANCR siRNA in cell growth and migration. Then, we detected downregulation of microRNA-204 (miR‑204), a suppressor of melanoma growth, in melanoma tissues and cell lines. We identified that miR‑204 was a direct target of BANCR and neurogenic locus notch homolog protein 2 (Notch2) was a direct target of miR‑204. BANCR may promote melanoma cell growth through inhibition of miR‑204, leading to the activation of Notch2 pathway. By tumorigenicity assay in BALB/c nude mice, we further demonstrated that BANCR knockdown inhibited tumor growth in vivo. Our results suggest the BANCR/miR‑204/Notch2 axis mediates melanoma cell proliferation and tumor progression.

KMT2A promotes melanoma cell growth by targeting hTERT signaling pathway

Melanoma is an aggressive cutaneous malignancy, illuminating the exact mechanisms and finding novel therapeutic targets are urgently needed. In this study, we identified KMT2A as a potential target, which promoted the growth of human melanoma cells. KMT2A knockdown significantly inhibited cell viability and cell migration and induced apoptosis, whereas KMT2A overexpression effectively promoted cell proliferation in various melanoma cell lines. Further study showed that KMT2A regulated melanoma cell growth by targeting the hTERT-dependent signal pathway. Knockdown of KMT2A markedly inhibited the promoter activity and expression of hTERT, and hTERT overexpression rescued the viability inhibition caused by KMT2A knockdown. Moreover, KMT2A knockdown suppressed tumorsphere formation and the expression of cancer stem cell markers, which was also reversed by hTERT overexpression. In addition, the results from a xenograft mouse model confirmed that KMT2A promoted melanoma growth via hTERT signaling. Finally, analyses of clinical samples demonstrated that the expression of KMT2A and hTERT were positively correlated in melanoma tumor tissues, and KMT2A high expression predicted poor prognosis in melanoma patients. Collectively, our results indicate that KMT2A promotes melanoma growth by activating the hTERT signaling, suggesting that the KMT2A/hTERT signaling pathway may be a potential therapeutic target for melanoma.

KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth

Melanoma is the most lethal form of skin cancer and treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors show only temporary benefit due the occurrence of resistance and immunotherapy is effective only in a subset of patients. To improve patient survival, there is a need to better understand molecular mechanisms that drive melanoma growth and operate downstream of the mitogen activated protein kinase (MAPK) signaling. The Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that plays a critical role in embryonic development, stemness and cancer, where it can act either as oncogene or tumor suppressor. KLF4 is highly expressed in post-mitotic epidermal cells, but its role in melanoma remains unknown. Here, we address the function of KLF4 in melanoma and its interaction with the MAPK signaling pathway. We find that KLF4 is highly expressed in a subset of human melanomas. Ectopic expression of KLF4 enhances melanoma cell growth by decreasing apoptosis. Conversely, knock-down of KLF4 reduces melanoma cell proliferation and induces cell death. In addition, depletion of KLF4 reduces melanoma xenograft growth in vivo. We find that the RAS/RAF/MEK/ERK signaling positively modulates KLF4 expression through the transcription factor E2F1, which directly binds to KLF4 promoter. Overall, our data demonstrate the pro-tumorigenic role of KLF4 in melanoma and uncover a novel ERK1/2-E2F1-KLF4 axis. These findings identify KLF4 as a possible new molecular target for designing novel therapeutic treatments to control melanoma growth.

Sildenafil Potentiates a cGMP-Dependent Pathway to Promote Melanoma Growth.

Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.

Clinical significance of long noncoding RNA SPRY4-IT1 in melanoma patients.

Long noncoding RNA SPRY4‐IT1 has been reported to promote melanoma cell growth and invasion, and to block apoptosis. The purpose of this study was to investigate the clinical significance of SPRY4‐IT1 in patients with malignant melanoma. The relative expression levels of SPRY4‐IT1 were measured in plasma samples from 70 patients and 79 healthy controls by quantitative reverse transcriptase polymerase chain reaction. SPRY4‐IT1 expression is high in melanoma patients but low in healthy controls, and is closely associated with tumor site and tumor stage. Elevated SPRY4‐IT1 significantly reduces overall survival rates of patients and is considered as an independent prognostic factor in patients with melanoma. The prognostic nomogram shows a good prediction of the probability of 5‐year overall survival of patients with melanoma (c‐index: 0.72). The calibration curve for the probability of survival presents good agreement between actual outcomes and predictive consequences. In summary, SPRY4‐IT1 may be a potential prognostic marker and a potential therapeutic target.

Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.

CEACAM1 Promotes Melanoma Cell Growth through Sox-2

The prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies.

Abstract 1743: SOX10 is required for cell cycle regulation and melanomagenesis.

Abstract The transcription factor SOX10 is essential for proper survival and differentiation of neural crest cell lineages, and plays an important role in the establishment and maintenance of melanocytes. SOX10 is also highly expressed in melanoma tumors, however its role in the progression of the disease is not yet understood. Here, we report that melanoma tumor cell lines require wild-type SOX10 expression for proliferation, and detail the rescue of melanoma in the metabotropic glutamate receptor 1 (Grm1Tg) transgenic mouse by Sox10 haploinsufficiency. Stable SOX10 knockdown in human melanoma cells results in arrested growth, altered cellular morphology, and senescence. Cells with a stable loss of SOX10 are arrested in the G1 phase of the cell cycle with elevated cyclin-dependent kinase inhibitor 1A and 1B (p21 and p27) expression. SOX10 loss also results in hypophosphorylation of retinoblastoma protein (RB) and a reduction in the expression of its binding partner E2F1. Previously, the RB-E2F1 pathway has been shown to play a central role in cell cycle regulation, and here we demonstrate that SOX10 expression is required for the maintenance of the RB-E2F1 pathway to promote melanoma cell growth. One of the main events driving cellular transformation is the dysregulation of the cell cycle, therefore the role that SOX10 has in maintaining this process provides great promise for targeted interventions. Citation Format: Julia C. Cronin, Dawn E. Watkins-Chow, Art Incao, Joanne H. Hasskamp, Nicola Schönewolf, Nicholas K. Hayward, Boris C. Bastian, Reinhard Dummer, Stacie K. Loftus, William J. Pavan. SOX10 is required for cell cycle regulation and melanomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2013-1743

Changes in oligosaccharide chains of autoantibodies to GRP78 expressed during progression of malignant melanoma stimulate melanoma cell growth and survival

A correlation between expression of the glucose-regulated protein of 78 kDa (GRP78) in malignant melanoma tumors and poor patient survival is well established. In this study, in addition to demonstrating the expression of GRP78 in tumor tissue, we investigated the immune response against GRP78 in a group of patients with different progression stages of malignant melanoma. Furthermore, we analyzed the glycosylation status of GRP78 immunoglobulin (Ig) G autoantibodies at these stages and evaluated their capacities to affect the protein B-dependent protein kinase signaling pathway and unfolded protein response signaling mechanisms, all known to promote malignant melanoma cell proliferation and survival. We found that progression of disease correlates not only with enhanced expression of GRP78 in the tumor but also with an increase in GRP78 autoantibody serum titers in these patients. We also found that the glycosylation status of anti-GRP78 IgG changes as the disease progresses. The anti-GRP78 IgG is abnormally glycosylated in the Fc region and asymmetrically glycosylated in the Fab region. We demonstrate that hyperglycosylated anti-GRP78 IgGs stimulate cell proliferation through protein B-dependent protein kinase signaling pathways. They also mimic the effects of α2-macroglobulin on the upregulation of GRP78 and X-box binding protein 1, activating transcription factor 6 α, and serine/threonine-protein kinase/endoribonuclease precursor α as endoplasmic reticulum stress biomarkers and show no effect on expression or activation of caspases 3, 9, or 12. In conclusion, the anti-GRP78 IgG autoantibodies downregulate apoptosis and activate unfolded protein response mechanisms, which are essential to promote melanoma cell growth and survival.

Abstract 1075: Cul1 Expression Is Increased in Early Stages of Human Melanoma

Abstract As the largest family of ubiquitin-protein E3 ligases, the SCF (Skp1/Cullin/Rbx1/F-box protein) complexes ubiquitinate a broad range of proteins involved in cell cycle progression, signal transduction and transcription. Cullin1 (Cul1) serves as a rigid scaffold in SCF complex for Rbx1, Skp1 and F-box protein subunits assembly and aberrant expression of Cul1 is involved in dysfunction of SCF E3 ligases. To investigate the role of Cul1 in the development of melanoma, we examined the expression of Cul1 in melanocytic lesions at different stages and analyzed the correlation between Cul1 expression and clinicopathologic parameters by tissue microarray and immunohistochemistry. The result showed that Cul1 expression was significant increased in primary and metastatic melanoma compared with dysplastic nevi, while there was no significant difference of Cul1 expression between primary and metastatic melanoma, which suggested that Cul1 plays an important role in the initiation stage of melanoma development. We found knockdown of Cul1 inhibited melanoma cell growth and overexpression of Cul1 promoted melanoma cell growth through regulating CDK inhibitor p27Kip expression. Knockdown of Cul1 abrogated Skp2-induced p27 degradation in melanoma cells. These results suggested that Cul1 is involved in cell cycle regulation of melanoma cells via Cul1-dependent ubiquitination and degradation of the p27kip1 by SCFSkp2 complex. In conclusion, our data indicate that Cul1 may serve as a potential marker for human melanoma initiation and early diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1075.