Promotes Melanoma Growth Research Articles

Regulation of Cancer Stem Cell Self-Renewal by HOXB9 Antagonizes Endoplasmic Reticulum Stress-Induced Melanoma Cell Apoptosis via the miR-765–FOXA2 Axis

Adaptation to endoplasmic reticulum (ER) stress has been indicated as a driver of malignancy and resistance to therapy in human melanoma. However, the relationship between cancer stem cells and adaptation to ER stress remains unclear. Here, we show that the ratio of cancer stem cells is increased in ER stress-resistant melanoma cells, which inhibit ER stress-induced apoptosis and promote tumorigenesis. Further mechanistic studies showed that HOXB9 triggered by ER stress favors cancer stem cell self-renewal and enhances ER stress resistance. HOXB9 directly binds to the promoter of microRNA-765 and facilitates its transcription, which in turn targets FOXA2, resulting in a FOXA2 decrease and cancer stem cell increase. Additionally, an increase in HOXB9 promotes melanoma growth and inhibits cell apoptosis in a mouse xenograft model. Elevated HOXB9 is found in human melanoma tissues, which is associated with microRNA-765 up-regulation and FOXA2 decreases. Thus, our data showed that the HOXB9-dependent, microRNA-765-mediated FOXA2 pathway contributes to the survival of melanoma under ER stress by maintaining the properties of cancer stem cells.

MELK Promotes Melanoma Growth by Stimulating the NF-κB Pathway

Melanoma accounts for more than 80% of skin cancer-related deaths, and current therapies provide only short-term benefit to patients. Here, we show in melanoma cells that maternal embryonic leucine zipper kinase (MELK) is transcriptionally upregulated by the MAPK pathway via transcription factor E2F1. MELK knockdown or pharmacological inhibition blocked melanoma growth and enhanced the effectiveness of BRAFV600E inhibitor against melanoma cells. To identify mediators of MELK function, we performed stable isotope labeling with amino acids in cell culture (SILAC) and identified 469 proteins that had downregulated phosphorylation after MELK inhibition. Of these proteins, 139 were previously reported as substrates of BRAF or MEK, demonstrating that MELK is an important downstream mediator of the MAPK pathway. Furthermore, we show that MELK promotes melanoma growth byactivating NF-κB pathway activity via Sequestosome 1 (SQSTM1/p62). Altogether, these results underpin an important role for MELK in melanoma growth downstream of the MAPK pathway.

The Tumor-Promoting Role of TRIP4 in Melanoma Progression and its Involvement in Response to BRAF-Targeted Therapy

TRIP4 was identified as having a proliferation promoting effect in melanoma cells based on small interfering RNA library screening, however, its precise function in melanoma progression is completely unknown. Here, we explored the carcinogenic role of TRIP4 in melanoma. The high expression of TRIP4 was observed in human melanoma cells and tissues. Its knockdown suppressed melanoma progression invitro and invivo, including melanoma cell proliferation, migration, and invasion inhibition and apoptosis induction. Further mechanistic analysis showed that TRIP4 promoted melanoma growth through modulation of COX-2 and iNOS expression partially by activating NF-κB signaling indirectly and partially by the direct anchoring of itself at COX-2 and iNOS promoter via synergy with p300. TRIP4 was confirmed to regulate the sensitivity to anti-BRAF targeted agents in BRAF-mutant human melanoma cells and xenografts. In addition, clinical data showed that high expression of TRIP4 was positively correlated with increased expression of COX-2 and iNOS and predicted poor prognosis in a cohort of 100 melanoma patients. Collectively, these results show a pro-tumorigenic role of TRIP4, provide an insight into the mechanism of TRIP4 as a candidate therapeutic target, and suggest the potential of TRIP4 and BRAF dual targeting as an effective therapeutic strategy for melanoma.

Paracrine Activin-A Signaling Promotes Melanoma Growth and Metastasis through Immune Evasion

The secreted growth factor Activin-A of the transforming growth factor β family and its receptors can promote or inhibit several cancer hallmarks including tumor cell proliferation and differentiation, vascularization, lymphangiogenesis and inflammation. However, a role in immune evasion and its relationship with tumor-induced muscle wasting and tumor vascularization, and the relative contributions of autocrine versus paracrine Activin signaling remain to be evaluated. To address this, we compared the effects of truncated soluble Activin receptor IIB as a ligand trap, or constitutively active mutant type IB receptor versus secreted Activin-A or the related ligand Nodal in mouse and human melanoma cell lines and tumor grafts. We found that although cell-autonomous receptor activation arrested tumor cell proliferation, Activin-A secretion stimulated melanoma cell dedifferentiation and tumor vascularization by functional blood vessels, and it increased primary and metastatic tumor burden and muscle wasting. Importantly, in mice with impaired adaptive immunity, the tumor-promoting effect of Activin-A was lost despite sustained vascularization and cachexia, suggesting that Activin-A promotes melanoma progression by inhibiting antitumor immunity. Paracrine Activin-A signaling emerges as a potential target for personalized therapies, both to reduce cachexia and to enhance the efficacy of immunotherapies.

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.

Abstract 374: KMT2A promotes tumor growth by activating hTERT and CBP signaling and predicts poor prognosis in human melanoma

Abstract Melanoma is an aggressive type of cutaneous malignancy. Although the inhibitors targeting BRAF and/or MEK pathways provide a therapeutic option for non-resectable melanoma driven by BRAF mutation, melanoma, especially metastatic melanoma, has still become one of the most threatening malignancies. Thus, identifying the exact molecular mechanisms involved in melanoma growth and discovering the novel targets for melanoma therapy is urgently needed. In this study, we screened a siRNA library targeting 6024 human genes in human melanoma cells and identified KMT2A as a potential therapeutic target for melanoma. KMT2A was highly expressed in melanoma cell lines and tumor tissues of melanoma patients. Knockdown of KMT2A by siRNA or shRNA significantly inhibited cell viability and colony formation, whereas exogenous expression of KMT2A effectively promoted cell growth in various melanoma cell lines. Further mechanism studies showed that the KMT2A-mediated regulation of melanoma growth was through targeting the hTERT and CBP signaling. Knockdown of KMT2A significantly inhibited hTERT promoter activity and protein expression and attenuated telomerase activity. Overexpression of hTERT rescued the KMT2A knockdown-mediated melanoma cell growth. By contrast, exogenous expression of KMT2A activated hTERT transcription and expression and increased telomerase activity in melanoma cells. Moreover, we found that KMT2A promoted hTERT expression and melanoma cell growth by cooperating with the transcriptional co-activator CBP, which interacted with and acetylated KMT2A. Inhibition of CBP by siRNA or a CBP-specific inhibitor suppressed the acetylation of KMT2A, abrogated the binding of KMT2A on the hTERT promoter and down-regulated hTERT expression, thereby inhibiting the growth of melanoma cells. Conversely, overexpression of CBP increased KMT2A acetylation and the binding to hTERT promoter, resulting in the promotion of hTERT expression and cell growth. The in vivo studies also showed that KMT2A promoted melanoma growth by activating the hTERT signaling in a xenograft tumor mouse model. Furthermore, the analyses for the clinical samples demonstrated that KMT2A expression was positively correlated with hTERT in tumor tissues of melanoma patients, and the high expression of both KMT2A and hTERT was associated with the worse clinical TNM staging and poor prognosis in melanoma patients. Taken together, our results indicate that KMT2A promotes melanoma growth by activating hTERT and CBP signaling in human melanoma. Our study therefore provides new insights into understanding the regulatory mechanism of melanoma growth and suggests that the KMT2A/CBP/hTERT signaling may be a potential therapeutic target for human melanoma. Funding support: This work was supported by the funds from the National Natural Science Foundation of China (81071687, 81272195), the State “973 Program” of China (2014CB542005). Citation Format: Changlin Zhang, Ranran Tang, Kefang Zhang, Wenlin Huang, Wuguo Deng. KMT2A promotes tumor growth by activating hTERT and CBP signaling and predicts poor prognosis in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2017-374

Abstract 3048: A noncoding function of TYRP1 mRNA promotes melanoma growth

Abstract RNAs competition to bind miRNA has been proposed to influence biological systems whether these RNAs are implicated in diseases onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly governs cell proliferation by sequestering miR-16 on non-canonical miRNA response elements (MREs). Consequently, sequestered miR-16 is no longer able to repress its mRNA targets such as RAB17, which we show is involved in melanoma cell proliferation and invasion. Restoration of miR-16 tumor suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression levels. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo using small oligonucleotides that mask miR-16 binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic noncoding function to TYRP1 mRNA and underscore miRNA displacement as a new antimelanoma targeted therapeutic approach. Citation Format: Marie-Dominique Galibert, David Gilot, Mélodie Migault, Laura Bachelot, Fabrice Journé, Aljosja Rogiers, Emmanuelle Donnou-Fournet, Ariane Mogha, Nicolas Mouchet, Marie-Laure Pinel-Marie, Bernard Mari, Tristan Montier, Sébastien Corre, Arthur Gautron, Florian Rambow, Petra El Hajj, Rania Ben Jouira, Sophie Tartare-Deckert, Jean-Christophe Marine, Brice Felden, Ghanem Ghanem. A noncoding function of TYRP1 mRNA promotes melanoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3048. doi:10.1158/1538-7445.AM2017-3048

NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma.

Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to IL-1β-mediated tumorigenesis in human melanoma. Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC, caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction. We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis. NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines. Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm. Knocking down NLRP1 revealed a tumor promoting property of NLRP1 both in vitro and in vivo. Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion, and NF-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma. However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death. Knocking down NLRP1 increased caspase-2, -9, and -3/7 activities and promoted apoptosis in human melanoma cells. Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases. Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9, and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation and apoptosis via interaction with caspase-2 and -9. In summary, we showed that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways. Our study demonstrates a tumor-promoting role of NLRP1 in cancer cells.

Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth.

Senescent cells within the tumor microenvironment (TME) adopt a proinflammatory, senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression, and therapeutic resistance. Here, exposure to palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA damage-independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure of nontransformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined melanoma cells (i.e., Ras mutant, Braf mutant, and Ras/Braf wild-type). When cultured in the presence of CDK4/6i-induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative responses. However, in vivo, PD-0332991-treated fibroblasts enhanced the growth of all melanoma lines tested and promoted the recruitment of Gr-1-positive immune cells. These data indicate that prolonged CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such senescent cells suppress the antitumor immune response and promote melanoma growth in immunocompetent, in vivo models.Implications: The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences that should be considered in clinical trials aiming to treat melanoma and other cancer types. Mol Cancer Res; 15(3); 237-49. ©2016 AACR.

Highlights of This Issue

In the current study, Williams and colleagues develop a proximity ligation assay (PLA) to measure the activity of Bcl-2 family members in situ. The team assesses Bcl-2 family inhibition in estrogen receptor-positive (ER+) breast cancers, finding poor response to ABT-263, a dual Bcl-2/Bcl-xL inhibitor, due to acute upregulation of a related factor, Mcl-1. Breast cancers from the clinic were determined to express higher transcript levels of the MCL1 gene, and were more frequently amplified, than other Bcl-2 members. Mcl-1 knockdown increases tumor cell death alone and in combination with ABT-263. Importantly, MCL1 transcript levels predict sensitivity to Bcl-2/Bcl-xL blockade, a timely finding as inhibitors of the Bcl-2 family enter clinical trials in breast cancer.Cyclin-dependent kinase 4/6 inhibitors (CDKi's) block tumor proliferation by mimicking p16INK4a function and are the subject of ongoing clinical trials. Similar to prolonged endogenous p16INK4a expression, extended CDK4/6i treatment promotes senescence in normal, proliferating cell lines. Senescence is associated with decreased regenerative capacity, age-related disease and cytokine secretion (i.e., SASP). Here, Guan and colleagues establish that primary fibroblasts, coaxed into senescence by prolonged CDK4/6i treatment, produce a robust cytokine profile/SASP that limits the anti-tumor immune response and promotes melanoma growth in immunocompetent models. These data reveal that the effects of CDK4/6i treatment on primary cells hinders therapeutic efficacy and promotes long-term, gerontogenic consequences.Mutations in the PIK3CA gene, activating phosphoinositide-3 kinase (PI3K), are commonly observed in colorectal cancer. Unfortunately, attempts at targeting this signaling cascade in colon cancer have resulted in significant therapeutic resistance. To investigate therapeutic strategies for this subtype, APC and PIK3CA mutant genetically engineered mice and spheroid cultures were generated. Using these model systems, dual PI3K/mTOR inhibition was discovered to induce treatment responses in spheroid cultures and in vivo. These results identify dual PI3K/mTOR inhibitors as a potential treatment strategy for patients with PIK3CA mutant colorectal cancer and deserve further clinical investigation.Previously we have shown that HER2+ breast cancers are susceptible to PARP inhibition (PARPi) independent of a DNA repair defect, and express elevated levels of PARP1. Now, Wielgos and colleagues discover that HER2 regulates PARP1 expression through suppression of the let-7a miRNA. Mechanistically, let-7a regulates PARP1 at the PARP1 3′-UTR. In human breast tumors, HER2 status correlates with high PARP1 protein and low let-7a. Lastly, introducing let-7a into HER2+ breast cancer cells induces cytotoxicity to a similar extent as the PARP inhibitor veliparib. Collectively, these findings demonstrate the potential of let-7a as a therapeutic strategy to target PARP1, and that baseline levels of let-7a and PARP1 are prospective biomarkers for PARPi sensitivity.

Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

Tumour hypoxia promotes melanoma growth and metastasis via High Mobility Group Box-1 and M2-like macrophages.

Hypoxia is a hallmark of cancer that is strongly associated with invasion, metastasis, resistance to therapy and poor clinical outcome. Tumour hypoxia affects immune responses and promotes the accumulation of macrophages in the tumour microenvironment. However, the signals linking tumour hypoxia to tumour-associated macrophage recruitment and tumour promotion are incompletely understood. Here we show that the damage-associated molecular pattern High-Mobility Group Box 1 protein (HMGB1) is released by melanoma tumour cells as a consequence of hypoxia and promotes M2-like tumour-associated macrophage accumulation and an IL-10 rich milieu within the tumour. Furthermore, we demonstrate that HMGB1 drives IL-10 production in M2-like macrophages by selectively signalling through the Receptor for Advanced Glycation End products (RAGE). Finally, we show that HMGB1 has an important role in murine B16 melanoma growth and metastasis, whereas in humans its serum concentration is significantly increased in metastatic melanoma. Collectively, our findings identify a mechanism by which hypoxia affects tumour growth and metastasis in melanoma and depict HMGB1 as a potential therapeutic target.

Abstract 2827: PD0332991-induced stromal senescence promotes melanoma growth in vivo

Abstract Cellular senescence, a process in which cells permanently exit the cell cycle, has pleiotropic biological effects. Several lines of evidence suggest that senescent cells within the tumor microenvironment can elicit a pro-inflammatory secretome that promotes cancer initiation and progression. This proinflammatory phenotype differs amongst senescent cells triggered by distinct stimuli; therefore, multiple forms of senescent cells likely exist within the tumor stroma during cancer evolution and treatment. Here, we sought to determine how stromal senescence initiated by clinically relevant treatments would influence melanoma growth. To generate senescent stromal populations, we treated presenescent MEFs (p4) with ultraviolet light (UV), Mitomycin C (MMC), or a CDK4/6 inhibitor (CDK4/6i). In contrast to p4 fibroblasts, cells treated with UV, MMC or CDK4/6i exhibited cell cycle arrest, increased SA-β-gal positivity and elevated p16INK4a expression. Pronounced 53BP1 and γH2AX foci were only observed in the UV and MMC treated cells, suggesting the CDK4/6i treatment did not induce DNA damage. Profiling of gene expression using a TaqMan mouse inflammation expression panel revealed distinctions amongst the 4 fibroblast lines. Of the senescent cell populations, CDK4/6i fibroblasts showed the highest number of elevated pro-inflammatory transcripts. To define the in vitro paracrine growth effects induced by stromal senescence, melanoma cell lines harboring either Braf600E, Ras, or unknown (wild-type) driver were co-cultured with each fibroblast population. In general, melanoma cells grew similarly on senescent and non-senescent fibroblasts; however, tumor cell growth was genotype-dependent. Notably, CDK4/6i fibroblasts never stimulated additional tumor cell growth in vitro. In contrast, syngeneic transplantations of fibroblasts and tumor cells into immune competent mice revealed that all senescent fibroblasts stimulated tumor growth in vivo. However, whether senescent and non-senescent fibroblasts promoted tumor growth at the same rate was genotype-dependent. Since this is the first study to examine the paracrine effects of senescent stromal cells using an immune competent mouse, we performed IHC for CD45 to assess whether immune cell recruitment differs in the presence of senescent stromal fibroblasts. No increase in CD45 positive infiltrates was observed in the melanoma lines co-injected with CDK4/6i fibroblasts. Together, this work reveals that melanoma growth in response to senescent stromal fibroblasts is genotype dependent, and that this stromal promotion of cancer growth can be accurately assessed only in the context of an immune competent, syngeneic host. Moreover, these data suggest that stromal senescence induced by emerging CDK4/6 inhibitor therapies and/or regimens combining chemotherapy/radiation with immune checkpoint blockade may also promote tumor growth through the paracrine effects of senescent bystanders. Citation Format: Xiangnan Guan, Kyle Lapak, Rebecca Hennessey, Christin Burd. PD0332991-induced stromal senescence promotes melanoma growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2827.

624 MALT1 acts though JNK/c-Jun to promote melanoma growth and metastasis

624 MALT1 acts though JNK/c-Jun to promote melanoma growth and metastasis

Neuropilin-2 promotes melanoma growth and progression in vivo.

Tumor cell interactions with their microenvironment, and neighboring endothelial cells in particular, are critical for tumor cell survival and the metastatic process. Within the spectrum of tumors, melanomas are notorious for their ability to metastasize at a relatively early stage of development; however, little is known about the molecular pathways mediating this process. We recently performed a screen to assess critical mediators of melanoma metastasis by evaluating melanoma-endothelial cell communication. Neuropilin-2 (NRP2), a cell surface receptor involved in angiogenesis and axonal guidance, was found to be an important mediator of melanoma-endothelial cell cross-talk in these studies. Here we seek to further define the role of NRP2 in melanoma growth and progression. We use stable gene silencing of NRP2 in melanomas from varying stages of tumor progression to define the role of NRP2 in melanoma growth, migration, invasion, and metastasis. We found that NRP2 gene silencing in metastatic melanoma cell lines inhibited tumor cell growth in vitro; furthermore, knockdown of NRP2 expression in the metastatic melanoma cell line 1205Lu significantly inhibited in-vivo tumor growth and metastasis. We conclude that NRP2 plays an important role in mediating melanoma growth and metastasis and suggest that targeting this cell surface molecule may represent a significant therapeutic strategy for patients diagnosed with aggressive forms of melanoma.

High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6.

The impact of metabolic stress induced by obesity on the bone marrow melanoma niche is largely unknown. Here we employed diet induced obese mice model, where mice received high-fat (HFD) or normal diet (ND) for 6 weeks before challenge with B16F10 melanoma cells. Tumor size, bone loss and osteoclasts numbers were assessed histologically in the tibial bones. For defining the molecular pathway, osteopontin knock-out mice, interleukin 6 neutralizing antibody or Janus kinase 2 inhibition were carried out in the same model. Mechanistic studies such as adipocyte-melanoma co-cultures for defining adipocyte induced changes of tumor cell proliferation and expression profiles were also performed. As results, HFD enhanced melanoma burden in bone by increasing tumor area and osteoclast numbers. This process was associated with higher numbers of bone marrow adipocytes expressing IL-6 in direct vicinity to tumor cells. Inhibition of IL-6 or of downstream JAK2 blocked HFD-induced tumor progression. Furthermore, the phenotypic changes of melanoma cells triggered macrophage and osteoclast accumulation accompanied by increased osteopontin expression. Osteopontin triggered osteoclastogenesis and also exerted a positive feedback loop to tumor cells, which was abrogated in its absence. Metabolic stress by HFD promotes melanoma growth in the bone marrow by an increase in bone marrow adipocytes and IL-6-JAK2-osteopontin mediated activation of tumor cells and osteoclast differentiation.

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.

MED27 promotes melanoma growth by targeting AKT/MAPK and NF-κB/iNOS signaling pathways

The inhibitors of BRAF and MEK targeting MAPK signaling pathway provide a comparatively effective therapeutic strategy for melanoma caused by BRAF mutation. However, melanoma, especially metastatic melanoma, has become one of the most threatening malignancies. Thus, the identification of exact molecular mechanisms and the key components involved in such mechanisms is urgently needed in order to provide new therapeutic options for patients with melanoma. Here, we identified MED27 as a potential melanoma target and explored its role and the associated molecular mechanism involved in melanoma progression. MED27 was found to be highly expressed in melanoma cells and tumor tissues. Its silencing led to melanoma cell proliferation inhibition, cell cycle arrest and apoptosis induction accompanied by the inactivation of PI3K/AKT and MAPK/ERK signaling and the activation of Bax/Cyto-C/Caspase-dependent apoptotic pathway. In addition, silencing of MED27 led to the decrease of iNOS expression through inhibiting the activation of a serial of upstream key proteins of NF-κB signaling pathway and the translocation of p50/p65 from cytoplasm to nucleus. MED27 was also found to be able to interact with NF-κB and p300 and to be acetylated by p300. Furthermore, the results in a xenograft tumor model indicated that melanoma progression was effectively suppressed by MED27 knockdown accompanied by the down-regulation of p-AKT, p-ERK, p-MEK1/2, MMP-9, Bcl-2 and iNOS expressions in the tumor tissues. Taken together, our study not only demonstrated the new function of MED27 as an oncogenic protein and the associated molecular mechanisms involved in melanoma progression, but also provided a possibility for the development of MED27 as a new anticancer target in melanoma therapy.

Leptin serves as angiogenic/mitogenic factor in melanoma tumor growth.

Background:Tumor development is angiogenesis dependent. There is evidence that leptin contributes to tumor growth. However, all the mechanisms by which leptin does this has not been clearly established. The objective of the present study was to test the hypothesis that leptin enhances melanoma tumor growth through inducing angiogenesis and cell proliferation.Materials and Methods:We injected 2 × 106 B16F10 melanoma cells subcutaneously to 32 C57BL6 mice. The mice were randomly divided into four groups of eight animals, on day 8. Two groups received twice daily intraperitoneal (i.p.) injections of either phosphate buffered saline or recombinant murine leptin (1 μg/g initial body weight). Two groups received i.p. injections of either 9F8 an anti leptin receptor antibody or the control mouse IgG at 50 μg/injection every 3 consecutive days. By the end of the 2nd week, the animals were euthanized and blood samples and tumors were analyzed. Angiogenesis and proliferation were assessed by immunohistochemical staining for CD31 and Ki-67 respectively.Results:Tumors size, capillary density, plasma levels of vascular endothelial growth factor, and the number of Ki-67-positive stained cells were significantly more in the leptin than 9F8 and both control groups (P < 0.05).Conclusion:Taken together, our findings reinforce the idea that leptin acts as an angiogenic and mitogenic factor to promote melanoma growth.

Nrf2 Deficiency Promotes Melanoma Growth and Lung Metastasis.

The role of Nrf2, a key regulator of antioxidant and cytoprotective genes, in tumorigenesis remains controversial. Here we showed that Nrf2 deficiency led to increased local tumor growth in mice following subcutaneous injection of B16-F10 melanoma cells, as indicated by increased proportion of animals with locally palpable tumor mass and time-dependent increases in tumor volume at the injection site. In vivo bioluminescence imaging also revealed increased growth of melanoma in Nrf2-null mice as compared with wild-type mice. By using a highly sensitive bioluminometric assay, we further found that Nrf2 deficiency resulted in a remarkable increase in lung metastasis of B16-F10 melanoma cells as compared with wild-type mice. Taken together, the results of this short communication for the first time demonstrated that Nrf2 deficiency promoted melanoma growth and lung metastasis following subcutaneous inoculation of B16-F10 cells in mice.