Activation of protein kinase C modulates cell–cell and cell–substratum adhesion of a human colorectal carcinoma cell line and restores ‘normal’ epithelial morphology

Abnormal cell adhesion is an important contributing factor in invasion and metastasis. Here, we show that morphologically ‘normal’ cell–cell and cell–substratum adhesion can be restored to a poorly differentiated carcinoma cell line by activation of protein kinase C (PKC). This cell line, VACO 10MS, grows as multicellular aggregates loosely attached to the substratum. The phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, 7.5 nM) induces rapid adhesive changes with 2 components. First, within 15 min of TPA the cells become closely apposed, an event resembling the ‘compaction’ seen in the mouse early embryo. Next, over 2 hr, the cells spread, forming a monolayer. We show that compaction depends on extracellular calcium, E‐cadherin‐mediated adhesion and F‐actin but not on protein synthesis, microtubules or substratum adhesion. By contrast, cell spreading is independent of cadherin and extracellular Ca²⁺ but involves the formation of focal contacts containing αv integrin. TPA treatment causes rapid translocation of PKC‐α to the insoluble fraction. During compaction, actin‐ and PKC‐α‐containing lamellae form over the entire aggregate surface, those adjacent to the substratum appearing to initiate spreading. Compaction does not involve increased phosphorylation of the cadherin/catenin complex. We conclude that activation of PKC‐α restores ‘normal’ morphology to these poorly differentiated cells. Our results are of general interest in relation to the regulation of cell adhesion and, through further investigation, may lead to identification of novel targets for therapeutic suppression of invasion and metastasis. Int. J. Cancer 80:455–464, 1999. © 1999 Wiley‐Liss, Inc.     

CD44 variant‐dependent redox status regulation in liver fluke‐associated cholangiocarcinoma: A target for cholangiocarcinoma treatment

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine–glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38^MAPK (a major ROS target) expression in Opisthorchis viverrini‐induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho‐p38^MAPK signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative‐phospho‐p38^MAPK patients a significantly shorter survival rate than the low CD44v signal and positive‐phospho‐p38^MAPK patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT‐targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38^MAPK in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT‐targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system.     

Overexpression of Necl‐5 correlates with unfavorable prognosis in patients with lung adenocarcinoma

Nectin‐like molecule‐5 (Necl‐5) is an immunoglobulin (Ig)‐like molecule that is up‐regulated in many types of cancer cells. It was shown experimentally that Necl‐5 enhances cell migration, proliferation, and metastasis, but its clinical significance has not been documented. The aim of this study was to observe the expression of Necl‐5 in surgically resected primary lung adenocarcinomas and to investigate its clinical significance. A total of 63 surgically resected primary pulmonary adenocarcinoma tissues were investigated by immunohistochemistry for the expression of Necl‐5. The relationship between expression of Necl‐5 and clinicopathological features was analyzed, and the influence of Necl‐5 expression on outcomes in these patients was assessed. A strong expression of Necl‐5 by cancer cells was observed in 43 of the 63 tumors. The overexpression of Necl‐5 by cancer cells was significantly associated with lymph node metastasis (P = 0.0398), TNM staging (P = 0.0367), and the bronchioloalveolar carcinoma ratio of tumors (P = 0.0423). Furthermore, the disease‐free survival rate in patients with positive Necl‐5 overexpression was significantly lower than that in patients with negative Necl‐5 overexpression (P = 0.0004). Multivariate survival analysis revealed Necl‐5 expression to be an independent risk factor for an unfavorable outcome (P = 0.0294). Additionally, an analysis including only the stage I cases revealed that the disease‐free survival rate of the Necl‐5‐positive group was significantly lower than that of the Necl‐5‐negative group (P = 0.0192). These results indicate that Necl‐5 plays a role in mediating tumor cell invasion and that the overexpression of Necl‐5 in cancer cells has clinical significance for prognostic evaluation of patients with primary pulmonary adenocarcinoma. (Cancer Sci 2010; 101: 1326–1330)     

Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts

The phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat-1 and Rat-2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter-mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C (PKC) α, PKCδ, PKCϵ, and PKCζ but neither the mRNA nor the protein for PKCβ. When Rat-2 fibroblasts were stably transfected with an expression vector producing PKCβ, however, TPA treatment of these variants resulted in a 3.1-fold induction of stromelysin promoter-mediated luciferase activity compared with a 1.3-fold induction in parental Rat-2 cells (P < 0.002). Transient transfection of PKCϵ produced a small but significant increase in TPA-stimulation of both stromelysin- and collagenase-mediated gene expression. These results suggest that there are PKC isotype-specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression. © 1996 Wiley-Liss, Inc.     

Regulation of spindle and kinetochore‐associated protein 1 by antitumor miR‐10a‐5p in renal cell carcinoma

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA‐10a‐5p (miR‐10a‐5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR‐10a‐5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR‐10a‐5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR‐10a‐5p in RCC cell lines (786‐O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore‐associated protein 1 (SKA1) was identified as an antitumor miR‐10a‐5p target by genome‐based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI‐treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR‐10a‐5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR‐10a‐5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.     

Metastasis‐associated in colon cancer‐1 upregulation predicts a poor prognosis of gastric cancer,and promotes tumor cell proliferation and invasion

Metastasis‐associated in colon cancer‐1 (MACC1) is a newly identified oncogene, and little is known about its role in gastric cancer (GC). Our study was performed to investigate whether MACC1 influences the prognosis of GC patients and to explore the potential mechanisms involved. MACC1 expression was verified to be higher in GC tissues than in adjacent nontumorous tissues by Western blotting. A retrospective analysis of 361 GC patients (Stages I–IV) revealed that higher MACC1 expression was associated with more advanced disease, more frequent postoperative recurrence, more metastases and a higher mortality rate. The disease‐free survival of Stage I–III patients and overall survival of Stage‐IV patients were significantly worse when their tumors showed high MACC1 expression. To investigate the underlying mechanisms, MACC1 overexpression and downregulation were established in two GC cell lines (BGC‐823 and MKN‐28 cells). MACC1 overexpression significantly accelerated tumor growth and facilitated metastasis in athymic mice. MACC1 also promoted the proliferation, migration and invasion of both GC cell lines. Moreover, gastric MACC1 mRNA expression levels were significantly correlated with markers of the epithelial‐to‐mesenchymal transition (EMT) in patients with GC. MACC1 overexpression upregulated mesenchymal–epithelial transition factor and induced changes to markers of EMT, whereas silencing of MACC1 reversed all these changes. These findings provide some novel insights into the role of MACC1, a gene that contributes to a poor prognosis of GC by promoting tumor cell proliferation and invasion as well as the EMT.     

Breast cancer cell invasiveness: Correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER⁺) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER⁻ cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER⁺ cells, constitutive PKC activity and PKCα expression were very low as compared to ER⁻ cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER⁺ and ER⁻ cells could be due to the abnormal TPA regulation of PKCα observed in ER⁻ cells. Int. J. Cancer 75:750–756, 1998.© 1998 Wiley-Liss, Inc.     

Immunocytochemical evidence for phorbol ester-induced directional translocations of protein kinase C in HL60, K562, CHO, and E7SKS cells: possible role in differentiation

The effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the directions of protein kinase C (PKC) translocation in two leukemic cell lines (HL60 and K562) and two fibroblastic cell lines (CHO and E7SKS), related to their susceptibility to the differentiating effect of TPA, were examined. Immunocytochemical evidence indicated that TPA induced a redistribution (outward) of PKC to the plasma membrane in TPA-sensitive HL60 cells, whereas it caused a translocation (inward) of the enzyme to the nucleus or the perinuclear region in K562, CHO, and E7SKS cells, which are resistant to TPA in terms of cell growth and differentiation. Immunoblot analysis of the nuclear proteins from K562 cells revealed that TPA induced an increase in the amount of immunoreactive proteins. TPA, however, did not increase the amount of these immunoreactive species in nuclei isolated from CHO and E7SKS cells, indicating that the translocated PKC was associated only with perinuclear structures of the TPA-treated cells. It is suggested that directional redistribution of PKC to the plasma membrane, as opposed to the nuclear and perinuclear region, might represent an early event required for the TPA-induced differentiation and maturation of HL60 cells.     

Expression of galectin‐1 in carcinoma‐associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin β1

Increased expression of galectin‐1 (Gal‐1) in carcinoma‐associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal‐1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal‐1, and established a co‐culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal‐1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal‐1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal‐1 expression level. A set of cancer invasion‐associated genes were then chosen to identify the possible mechanism of Gal‐1‐induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal‐1 expression. Pre‐blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion‐promoting effect of CAFs with high Gal‐1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal‐1 and integrin β1 expression. Our results showed that high expression of Gal‐1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.     

Coding microsatellite instability analysis in microsatellite unstable small intestinal adenocarcinomas identifies MARCKS as a common target of inactivation

Approximately 15% of small intestinal adenocarcinomas show inactivation of DNA‐mismatch repair (MMR) and display high‐level microsatellite instability (MSI‐H). MSI‐H tumors progress as a result of mutations affecting coding microsatellites (coding microsatellite instability, cMSI) that may result in a functional inactivation of the encoded proteins and provide a selective growth advantage for the affected cell. To investigate the cMSI selection in small intestinal carcinogenesis 56 adenocarcinomas were tested for MSI. Eleven MSI‐H carcinomas (19.6%) were identified and subjected to cMSI analysis in 24 potentially tumor relevant genes. Mutation frequencies were similar to those observed in colorectal cancer (CRC). Beside high frequencies of cMSI in TGFβR2, ACVR2, and AIM2 we detected MARCKS mutations in 10 out of 11 (91%) tumors with a 30% share of biallelic mutations. Since little is known about MARCKS expression in the intestine, we analyzed MARCKS protein expression in 31 carcinomas. In non‐neoplastic mucosa, MARCKS was found to be expressed with a concentration gradient along the crypt–villus axis. In line with cMSI induced functional inactivation of MARCKS, 8 out of 11 MSI‐H adenocarcinomas showed regional or complete loss of the protein. In microsatellite stable (MSS) small bowel adenocarcinoma, loss of MARCKS expression was seen in 2 out of 20 tumors (10%). In conclusion, we herein present a cMSI profile of MSI‐H small intestinal adenocarcinomas identifying MARCKS as a frequent target of mutation. Loss of MARCKS protein expression suggests a significant role of MARCKS inactivation in the pathogenesis of small intestinal adenocarcinomas. © 2009 Wiley‐Liss, Inc.     

CENP‐50 is required for papilloma development in the two‐stage skin carcinogenesis model

CENP‐50/U is a component of the CENP‐O complex (CENP‐O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP‐50/U has been reported in many types of cancers. However, as Cenp‐50/U‐deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp‐50/U in skin carcinogenesis, we generated Cenp‐50/U conditional knockout (K14Cre^ER‐Cenp‐50/U^fl/fl) mice and subjected them to the 7,12‐dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early‐stage papillomas decreased in Cenp‐50/U‐deficient mice. In contrast, Cenp‐50/U‐deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA‐induced papillomas and carcinomas revealed that Cenp‐50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp‐50/U functions mainly in early papilloma development and it has little effect on malignant conversion.     

Prognostic value of phospho‐Akt in patients with non‐small cell lung carcinoma: A meta‐analysis

Previous studies have been inconsistent with respect to the reported associations between phospho‐Akt (p‐Akt) overexpression and lung cancer prognosis. In this study, we conducted a systematic review and meta‐analysis to assess the prognostic value of p‐Akt in patients with non‐small cell lung carcinoma (NSCLC). Relevant articles were identified by searching MEDLINE. Hazard risks (HRs) from individual studies were calculated and pooled by using a random‐effect model, and heterogeneity and publication bias analyses were also performed. Finally, 18 studies comprising 2,353 patients were included in the meta‐analysis. p‐Akt overexpression was associated with worse survival in NSCLC patients, and the pooled HRs for all the studies was 1.38 (95% confidence interval [CI]: 1.11–1.70; p < 0.01). After subgroup analysis, the association was strengthened in the surgery treatment group, with an HR of 1.44 (95% CI: 1.19–1.75; p < 0.01), while in the tyrosine kinase inhibitors treatment group, the statistical significance disappeared (HR: 1.22, 95% CI: 0.70–2.14; p = 0.48). The HR in cases of early stage disease (I–III) was 1.35 (95% CI: 1.08–1.69; p = 0.04); however, in cases of late stage disease (III–IV), the association became non‐significant (HR: 1.22, 95% CI: 0.64–2.33; p = 0.54). Our results suggest that there was a significantly inverse association between p‐Akt overexpression and the prognosis of NSCLC patients, and that this association appeared to be limited in early‐stage patients who underwent surgery.     

Significant role of Psf3 expression in non‐small‐cell lung cancer

The GINS complex associates with cell division cycle (Cdc) protein 45 and mini‐chromosome maintenance (Mcm) proteins 2–7 to form the Cdc45–Mcm–GINS (CMG) complex, which is essential for DNA duplication. One member of the GINS complex is Psf3. We previously found that increased Psf3 expression was strongly associated with poor survival in lung adenocarcinoma. Here, we investigated the role of Psf3 expression in non‐small‐cell lung cancer (NSCLC). We verified Psf3 expression in human NSCLC tissues (180 patients) and cell lines. Immunohistochemical analysis revealed that the overexpression of Psf3 was significantly associated with vessel invasion (P = 0.016), lymphatic invasion (P = 0.002), and pleural invasion (P = 0.036). The overall survival rate in patients with Psf3 overexpression was significantly lower than that in patients without Psf3 overexpression (P = 0.006). Multivariate survival analysis revealed Psf3 expression to be an independent risk factor for an unfavorable outcome (P = 0.049). A proximal ligation assay showed interactions between Psf3 and other CMG components (such as Mcm2 and Cdc45) in both NSCLC specimens and cell lines, indicating that Psf3 acted as the CMG complex, which could lead to excessive proliferation. Knockdown of Psf3 inhibited the proliferation of both cell lines by delaying the S phase, which revealed that Psf3 played an important role in cancer proliferation. Thus, Psf3 acted as the CMG complex, promoting excessive proliferation. These results suggest that Psf3 inhibition might be a therapeutic target for NSCLC with Psf3 overexpression.     

Increased transforming growth factor‐α levels in human colon carcinoma cell lines over‐expressing protein kinase C

Transforming growth factor‐α (TGF‐α) is synthesized as a membrane‐bound precursor protein, pro‐TGF‐α, that is converted to a soluble form by 2 endoproteolytic cleavages. Several factors have been implicated in the regulation of the second rate‐limiting step, including protein kinase C (PKC). Earlier results indicated a potential role for the conventional class of PKC isozymes in the observed increase in TGF‐α in the conditioned media of 2 human colon carcinoma cell lines. The present study addresses the potential role of specific PKC isozymes in this process using sense and anti‐sense expression vectors for PKC isozymes. Two human colon carcinoma cell lines, HCT 116 and GEO, were transfected with plasmids, leading to the over‐expression of PKC‐α, ‐βI or ‐βII; and the secretion of TGF‐α into the conditioned medium was determined. Over‐expression of either PKC‐βI or PKC‐βII in these cell lines enhanced the levels of TGF‐α in the media 2‐ to 5‐fold. Over‐expression of PKC‐α did not alter the amount of TGF‐α in the media to a significant extent. Transfection of HCT 116 cells with the anti‐sense PKC‐βI cDNA resulted in a reduction in PKC‐βI protein expression. This was accompanied by a decrease in the amount of TGF‐α in the conditioned media. Our results indicate that modulation of PKC‐β protein levels alters the amount of TGF‐α found in the conditioned media from these colon carcinoma cells. Int. J. Cancer 80:72–77, 1999. © 1999 Wiley‐Liss, Inc.     

EBAG9 is a tumor‐promoting and prognostic factor for bladder cancer

Upregulation of EBAG9 expression has been observed in several malignant tumors such as advanced breast and prostate cancers, indicating that EBAG9 may contribute to tumor proliferation. In the present study, we assess the role of EBAG9 in bladder cancer. We generated human bladder cancer EJ cells stably expressing FLAG‐tagged EBAG9 (EJ‐EBAG9) or empty vector (EJ‐vector), and investigated whether EBAG9 overexpression modulates cell growth and migration in vitro as well as the in vivo tumor formation of EJ transfectants in xenograft models of BALB/c nude mice. EBAG9 overexpression promoted EJ cell migration, while the effect of EBAG9 to cultured cell growth was rather minimal. Tumorigenic experiments in nude mice showed that the size of EJ‐EBAG9‐derived tumors was significantly larger than EJ‐vector‐derived tumors. Loss‐of‐function study for EBAG9 using small interfering RNA (siRNA) in xenografts with parental EJ cells showed that the intra‐tumoral injection of EBAG9 siRNA markedly reduced the EJ tumor formation compared with control siRNA. Furthermore, immunohistochemical study for EBAG9 expression was performed in 60 pathological bladder cancer specimens. Intense and diffuse cytoplasmic immunostaining was observed in 45% of the bladder cancer cases. Positive EBAG9 immunoreactivity was closely correlated with poor prognosis of the patients (p = 0.0001) and it was an independent prognostic predictor for disease‐specific survival in multivariate analysis (p = 0.003). Our results indicate that EBAG9 would be a crucial regulator of tumor progression and a potential prognostic marker for bladder cancer. © 2008 Wiley‐Liss, Inc.     

Growth of human melanocyte cultures supported by 12-O-tetradecanoylphorbol-13-acetate is mediated through protein kinase C activation.

To investigate the role of protein kinase C (PKC) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent growth of human melanocytes, we analyzed the effects of phorbol ester treatment on both PKC expression and growth control in these cells. We found that established cultures of normal melanocytes contain the PKC alpha, PKC beta, and PKC epsilon isoforms. The abilities of various phorbol ester compounds to stimulate DNA synthesis in these cultured melanocytes correlated with their known potencies for activation of PKC and tumor promotion. Dose-response studies revealed that the most effective TPA concentration for stimulation of DNA synthesis and growth of melanocytes (10 ng/ml TPA) also supported a relatively high level of PKC enzyme activity, increased membrane association of the PKC alpha and PKC epsilon isoforms, and led to a high level of phosphorylation of a major PKC substrate, the myristoylated alanine-rich C kinase substrate (MARCKS) protein. Melanocytes incubated for 48 h with TPA at a higher concentration (100 ng/ml TPA) exhibited suboptimal TPA-stimulated DNA synthesis (28% of maximal) and decreased phosphorylation of the MARCKS substrate protein (50% of maximal). Furthermore, treatment of melanocytes with 100 ng/ml TPA for 48 h resulted in a marked decrease in total PKC enzyme activity and the loss of expression of the PKC alpha and PKC epsilon isoforms in both the cytosol and membrane-bound fractions, when examined by immunoblot analysis. These results, taken together, suggest that continuous activation of PKC by TPA, rather than the loss of PKC due to TPA-induced down-regulation, is responsible for the growth-stimulatory effects of phorbol esters on normal human melanocytes. Additionally, the conditioned medium from TPA-treated human melanocytes stimulated DNA synthesis in quiescent melanocytes and human melanoma cells, thus suggesting that activation of the PKC signaling pathway in melanocytes leads to the production of an autocrine growth factor. These findings may be relevant to the autonomous growth of malignant melanomas.