Growth Of Melanoma Cells Research Articles

MiR-302a-3p suppresses melanoma cell progression via targeting METTL3

The miRNA-302 family plays a critical role in carcinogenesis. As an enzyme that regulates the N6-methyladenosine modification, methyltransferase-like 3 (METTL3) plays important roles in the development and progression of various tumours. However, the upstream regulatory mechanisms of METTL3 in melanoma have not yet been fully investigated. Herein, we investigated the functions of miR-302a-3p and its target RNA METTL3 on proliferation, apoptosis, and invasion of melanoma. Quantitative real-time PCR and immunofluorescence staining were used to measure the expression of METTL3 mRNA and protein level after transfection. miR-302a-3p expression was determined by quantitative reverse transcription-PCR. The cell proliferation, cell cycle progression, apoptosis, colony formation, migration, and cell invasion ability were determined using MTT assay, propidium iodide (PI) staining, Annexin V/PI flow cytometry, plate clone assay, and Transwell migration and invasion assays, respectively. Melanoma cell metastasis was also evaluated using an in vivo model. The effect of METTL3 on the phosphorylation of PI3K and AKT was measured with western blot analysis. Our results showed that miR-302a-3p was significantly downregulated in melanoma and exerted a tumour suppressive role against melanoma progression. We identified METTL3 as a direct target of miR-302a-3p in melanoma cells using bioinformatics analysis and luciferase assay. Furthermore, the enforced overexpression of METTL3 promoted the proliferation, cell cycle progression, cell invasion, migration, expression of epithelial-to-mesenchymal transition markers, and the PI3K-AKT signalling pathway as well as suppressed the apoptosis of melanoma cells. Meanwhile, silencing the expression of METTL3 with specific shRNA demonstrated reverse outcomes of the above phenotypes in melanoma cells. By rescue experiments, we found that the restoration of METTL3 expression in miR-302a-3p-overexpressing melanoma cells successfully recovered the miR-302a-3p-mediated melanoma suppression. The in vivo results also showed that miR-302a-3p substantially inhibited melanoma cell growth and metastasis. In summary, this study demonstrated that miR-302a-3p targets METTL3 and plays tumour suppressive roles in the proliferation, apoptosis, invasion, and migration of melanoma cells.

Abstract 2609: Hypoxia-mediated downregulation of GCNT2/I-antigen in metastatic melanoma accelerates disease progression and mortality

Abstract Metastatic melanoma is a lethal disease with a dismal 5-year survival rate. Thus, intense efforts to boost novel therapeutic strategies are underway to identify early detection of melanomas with a high propensity to metastasize. We recently discovered that the loss of cell surface glycan, I-antigen, corresponds with the transition of primary melanoma to metastatic melanoma. I-antigen or I-branched glycans are synthesized by β16, N-acetylglucosaminyltransferase 2 (GCNT2) and inversely correlate with the growth and signaling potential of metastatic melanoma cells. Moreover, compared with high GCNT2 expression in normal melanocytes, nevi, and early-stage primary melanomas, GCNT2 is conspicuously lost in metastatic melanomas. We anticipate the potential utilization of GCNT2 expression as a biomarker to predict melanoma metastasis. Further, metastasis and aggressive disease progression are key phenotypes of tumor-initiating cells (TIC), which are preferentially generated in areas of hypoxia. In the vertical growth phase of primary melanomas and melanoma metastases, the tumor microenvironment is typically hypoxic (1.5% oxygen). We hypothesize that the hypoxic microenvironment aids in metastatic melanoma progression through TIC generation and immune evasion, by downregulating GCNT2 and switching I-branched glycans to linear glycans. In this study, metastatic melanoma cells grown under hypoxic conditions had reduced GCNT2 and MITF with upregulated stem cell marker KLF4 expression. Importantly, in the in vivo TIC assay, we found significantly decreased tumor formation with increased GCNT2 expression while low GCNT2 levels enabled tumor formation even when 103 cells were injected in immunocompromised mice. Since TICs are thought to evade immune clearance, we investigated whether loss of GCNT2 increased TIC characteristics and also enabled immunosuppressive features. In human PBMC - metastatic melanoma co-cultures, there was an increase in T regulatory cell generation associated with low GCNT2 compared to high GCNT2 expression in melanoma cells, suggesting that loss of GCNT2 associates with increased TIC generation, tumor formation, and immunoevasion potential. Using melanoma patient specimens, immunohistochemical analysis of GCNT2 corresponded with a significant increase in mortality with the loss of GCNT2 staining. Altogether, these findings highlight GCNT2/I-branching not only as a biomarker of melanoma virulence but reveal malignancy-associated pathways functioning in parallel with loss of GCNT2/I-branching that could offer additional targets for the treatment of metastatic melanoma. Citation Format: Asmi Chakraborty, Mariana Perez, Norhan B. B Mohammed, Michael Wells, James S. Wilmott, John F. Thompson, Stuart M. Haslam, Wei Wang, Richard A. Scolyer, George F. Murphy, Charles J. Dimitroff. Hypoxia-mediated downregulation of GCNT2/I-antigen in metastatic melanoma accelerates disease progression and mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2609.

Abstract 2858: DEPDC1B functions as an oncogene in promoting melanoma growth and metastasis

Abstract Background: Melanoma is one of the most aggressive skin cancers due to its therapeutic resistance, and high capacity to metastasize as a late manifestation. The limited effectiveness of the current treatment regimen prompts us to identify new druggable targets in governing the systemic spread of melanoma. Despite the oncogenic role of DEPDC1B in different types of cancer, its functional importance in melanoma growth and metastasis are not fully revealed. Methods: The expression of DEPDC1B mRNA levels between skin cutaneous melanoma patients and normal samples were compared using TCGA database. Surgically procured tumor specimens from melanoma patients were obtained to investigate the protein levels of DEPDC1B expression by immunofluorescence. Melanoma cell lines WM266-4 and SK-MEL-28 were used for loss- and gain-of-function studies of DEPDC1B in AlamarBlue, EdU staining, colony formation and trans-well invasion assays. Subcutaneous and intravenous injection of melanoma cells treated with either DEPDC1B knockdown (KD) or DEPDC1B overexpression into immunodeficient mice were conducted to examine the effects on tumor growth and lung metastasis respectively. Results: The expression levels of DEPDC1B mRNA in melanoma biopsies were higher than normal samples and were negatively corelated with both overall survival and disease-free survival. Loss-of-function studies showed that DEPDC1B KD reduced the growth rate, proliferation capacity and colony formation ability of melanoma cells. Consistently, DEPDC1B KD cells gave rise to smaller xenograft tumors and fewer number of lung nodules. In contrast, DEPDC1B overexpression promoted melanoma cell proliferation, colony formation and invasion in vitro, and enhanced xenograft tumor growth and lung metastasis. Conclusion: The results obtained so far indicate that DEPDC1B acts as an oncogene in promoting melanoma cell growth and metastasis, suggesting a potential drug target in melanoma treatment. Citation Format: Feng Hu, Ki On Fong, May Pui Lai Cheung, Xintao Yang, Martin Cheung. DEPDC1B functions as an oncogene in promoting melanoma growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2858.

Abstract LB261: BRAF inhibitor reprograms cancer-associated fibroblasts to elicit matrix remodeling and therapeutic escape in melanoma

Abstract The tumor stroma and its cellular components are known to play important roles in tumor resilience towards treatment but remain to be urgently characterized in melanoma targeted therapy. By performing a paired-analysis of melanoma tissue samples collected from the patients before and after combined BRAF inhibitors (BRAFi)/MEK inhibitors (MEKi) treatment, we discovered that the number of intratumoral cancer-associated fibroblasts (CAFs) increased significantly in BRAFi/MEKi-treated melanoma stroma comparing with the samples before the therapy. Those intratumoral CAFs exhibit increased amounts of nuclear β-catenin under BRAFi/MEKi therapy. Further studies show that BRAFi not only leads to increased β-catenin nuclear accumulation in CAFs and enhances their biological properties, suggesting BRAFi induces a new resistant phenotype in CAFs. In both in vitro and in vivo models, depleting β-catenin in CAFs abolishes their ability to elicit melanoma cell growth and drug resistance. BRAFi induces BRAF and CRAF dimerization and subsequently the activation of MEK/ERK signaling in CAFs, leading to the inactivation of the β-catenin destruction complex. RNA-Seq data reveals that nuclear β-catenin is a major regulator of CAF phenotype. We identify matricellular protein periostin (POSTN) as a downstream effector of β-catenin. In melanoma, POSTN is uniquely expressed in CAFs, and BRAFi can specifically upregulate POSTN production in CAFs but not in melanoma cells. Recombinant POSTN contributes to melanoma cell BRAFi/MEKi resistance and can compensate for the loss of β-catenin in CAFs. By reactivating ERK pathway in melanoma cells through PI3K/AKT signaling, POSTN compensates for the loss of β-catenin in CAFs in conferring melanoma cell BRAFi/MEKi resistance. In summary, our data reveal a novel resistance mechanism in melanoma that is elicited by BRAFi-induced non-canonical nuclear β-catenin signaling in CAFs and reveal POSTN as an important matrix target to eliminate BRAFi/MEKi resistance in melanoma. Citation Format: Tianyi Liu, Linli Zhou, Yuhang Zhang. BRAF inhibitor reprograms cancer-associated fibroblasts to elicit matrix remodeling and therapeutic escape in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB261.

An In Vitro-In Vivo Evaluation of the Antiproliferative and Antiangiogenic Effect of Flavone Apigenin against SK-MEL-24 Human Melanoma Cell Line.

One of the most important class of natural compounds with successful preclinical results in the management of cancer is the flavonoids. Due to the plethora of biological activities, apigenin (4′,5,7 trihydroxyflavone) is a main representant of the flavone subclass. Although the antiproliferative and antiangiogenic effects of apigenin were studied on a significant number of human and murine melanoma cell lines, in order to complete the data existing in the literature, the aim of this study is to evaluate the in vitro effect of apigenin on SK-MEL-24 human melanoma cell line as well as in vivo on tumor angiogenesis using the aforementioned cell line on the chorioallantoic membrane assay. Results have shown that in the range of tested doses, the phytocompound presents significant antiproliferative, cytotoxic, and antimigratory potential at 30 μM, respectively, 60 μM. Moreover, the phytocompound in both tested concentrations limited melanoma cell growth and migration and induced a reduced angiogenic reaction limiting melanoma cell development.

The Biological Effectiveness and Medical Significance of Far Infrared Radiation (FIR)

The electromagnetic waves constitute different wavelengths of light from which Far infrared (FIR) is beneficial for living cells. Extensive studies and trials have been conducted over the last two decades in multidimensional biological domains to identify its unlimited health benefits. FIR radiations improve the microcirculation of the human body, stimulate cell growth, penetrate through skin tissues non-invasively, create intramolecular vibrations create an overall healthy metabolism, which ultimately affects overall improved cardiac and metabolic activity. This phenomenon is used to explore different pathological conditions to identify their significance in the medical field. In this review, we explored the biological effectiveness and the medical significance of Far infrared radiation (FIR) in murine melanoma Cell Growth, Lymphedema, airborne viruses, Cardiac diseases, Wound healing and burns, Autonomic Activities, Hemodialysis, Allergic Rhinitis, Aesthetic medicine, textiles, and other domains such as obesity and gut microbiota.

Inhibitory effect of Saccharomyces cerevisiae extract obtained through ultrasound-assisted extraction on melanoma cells

Although the immune enhancing effect of yeast has been widely reported, studies specifically investigating its effects on skin cancer are lacking. Therefore, this study aimed to develop a yeast extract capable of inhibiting melanoma cells using ultrasound technology, which can lyse the cell walls allowing subsequent rapid yeast extraction. To compare the extraction efficiency across different extraction methods, the total yield, as well as total glucan, α-glucan, and β-glucan yields were measured. Ultrasound-assisted extract of yeast (UAEY) was found to effectively inhibit melanoma cell growth and proliferation as well as the expression of cyclin D1 and c-myc, in vitro. Additionally, the extract reduced melanoma tumor volume and cyclin D1 levels in BALB/c nu/nu mice. The optimal extraction conditions were 0.2 M NaOH, 3 h, 70 °C, 20 kHz, and 800 W, resulting in an increased total extraction and β-glucan yields of 73.6% and 7.1%, respectively, compared with that achieved using a conventional chemical (0.5 M NaOH) extraction method. Taken together, the results of this study suggest that UAEY may represent an effective anti-skin cancer agent.

A carbazole compound, 9-ethyl-9H-carbazole-3-carbaldehyde, plays an antitumor function through reactivation of the p53 pathway in human melanoma cells

p53, the major tumor suppressor, is frequently mutated in many cancers, and up to 84% of human melanomas harbor wild-type p53, which is considered to be an ideal target for melanoma therapy. Here, we evaluated the antitumor activity of a carbazole derivative, 9-ethyl-9H-carbazole-3-carbaldehyde (ECCA), on melanoma cells. ECCA had a selectively strong inhibitory activity against the growth of BRAF-mutated and BRAF-wild-type melanoma cells but had little effect on normal human primary melanocytes. ECCA inhibited melanoma cell growth by increasing cell apoptosis, which was associated with the upregulation of caspase activities and was significantly abrogated by the addition of a caspase inhibitor. In vivo assays confirmed that ECCA suppressed melanoma growth by enhancing cell apoptosis and reducing cell proliferation, and importantly ECCA did not have any evident toxic effects on normal tissues. RNA-Seq analysis identified several pathways related to cell apoptosis that were affected by ECCA, notably, activation of the p53 signaling pathway. Biochemical assays demonstrated that ECCA enhanced the phosphorylation of p53 at Ser15 in melanoma cells harboring wild-type p53, and importantly, the knockdown or deletion of p53 in those cells counteracted the ECCA-induced apoptosis, as well as senescence. Further investigations revealed that ECCA enhanced the phosphorylation of p38-MAPK and c-Jun N-terminal kinase (JNK), and treatment with either a p38-MAPK or a JNK inhibitor rescued the cell growth inhibition elicited by ECCA, which depended on the expression of the p53 gene. Finally, the combination of ECCA with a BRAF inhibitor significantly enhanced the growth inhibition of melanoma cells. In summary, our study demonstrates that the carbazole derivative, ECCA, induces melanoma cell apoptosis and senescence through the activation of p53 to significantly and selectively suppress the growth of melanoma cells without affecting normal human melanocytes, suggesting its potential to develop a new drug for melanoma therapy.

Pectin Oligosaccharides Enhance α2,6-Sialylation Modification that Promotes Apoptosis of Bladder Cancer Cells by Targeting the Hedgehog Pathway.

Although pectin oligosaccharide (POS) can inhibit the growth and proliferation of gastric, colon, prostate, breast, melanoma, and leukemia cells, its effect on bladder cancer remains unknown. Therefore, screening and identification of factors associated with the sensitivity of bladder cancer to drugs and elucidation of their molecular mechanisms will help provide a theoretical basis for establishing postoperative systemic chemotherapy for patients with bladder cancer. We showed that POS promoted the apoptosis of bladder cancer cells, and this finding was consistent with enhanced α2,6-sialylation post-modification. Moreover, POS activated the Hedgehog pathway, the inhibition of which regulated the tumorigenicity of bladder cancer cells in vivo. These findings were consistent with our results in vitro. We conclude that POS promotes the apoptosis of bladder cancer and offers new insights and evidence for the development of individualized treatment strategies. Schema of molecular events underlying POS-induced inhibition of bladder cancer cell proliferation.

ETS1 promoted cell growth, metastasis and epithelial-mesenchymal transition process in melanoma by regulating miR-16-mediated SOX4 expression.

Melanoma is a malignant tumor with high metastasis and mortality. Epithelial-mesenchymal transition (EMT) was reported to be involved in the growth and metastasis of melanoma. To investigate these sections further, we showed that E26 transformation specific 1 (ETS1) could regulate growth, metastasis and EMT process of melanoma by regulating microRNA(miR)-16/SRY-related HMG box (SOX) 4 expression. MiR-16, ETS1, SOX4 and nuclear factor κB (NF-κB) expression levels in melanoma cells were examined using qPCR. ETS1, SOX4, EMT-related proteins and NF-κB signaling pathway-related proteins were examined using western blot. Cell counting kit-8 assay, transwell assay were applied to evaluate the cell proliferation, migration and invasion of melanoma cells, respectively. Besides, a dual-luciferase reporter assay was employed to verify the binding relationship between ETS1 and miR-16, miR-16 and SOX4, miR-16 and NF-κB1. We showed that ETS1 and SOX4 were upregulated in melanoma cells, while miR-16 was downregulated. MiR-16 overexpression suppressed growth, metastasis and EMT process of melanoma. We found ETS1 could bind to the promoter region of miR-16 and inhibited its transcription. ETS1 silence could inhibit growth, metastasis and EMT process of melanoma, and inhibition of miR-16 could reverse the effects. Besides, miR-16 is directly bound to SOX4 and downregulated its expression. Rescued experiments confirmed that SOX4 overexpression abolished the inhibition effect of miR-16 mimics on growth, metastasis and EMT process of melanoma. Finally, NF-κB1 as the target of miR-16 mediated downstream biological responses. ETS1 activated NF-κB signaling pathway through miR-16 via targeting SOX4, thus promoting growth, metastasis and EMT of melanoma.

In-Vitro Effects of Active Vitamin-D3 and Vitamin-A on Human Melanoma (BLM and1205Lu) Cell Growth

Though UV ray in the Sun light is essential for vitamin-D3 formation, yet UV rays can lead to skin cancer. Lack of vitamin-D3 also can lead to cancer. Hence, we decided to study the effect of vitamin-D3 on human melanoma cell models. Our aims were to study the in-vitro effects of vitamin-D3 and vitamin-A on human melanoma (BLM, 1205Lu) cell growth, as vitamins D3 and A are important for a healthy skin. Initially dose-curve (100 nM to 100 μM concentration) study was carried out with vitamin-D3 and vitamin-A (retinoic acid) on BLM cells to determine the optimal concentrations of vitamins-D3 and A for co-incubation with progesterone (50 μM) and RU-486 (50 μM). Supernatants from the treated cells were subjected to Elisarray. Vitamin-D3 and vitamin-A showed a dose-dependent decrease in cell growth. Based on the dose-curve, it was decided to use 25 μM (at 57% cell growth) of vitamin-D3 and 50 μM (at 55% cell growth) of vitamin-A for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of BLM cell growth (20%). Similarly co-incubation of vitamin-D3 with progesterone (33% cell growth) and with RU-486 (28% cell growth) as well as co-incubation of vitamin-A with progesterone (31% cell growth) and RU-486 (18% cell growth) showed an additive effect on the decrease of BLM cell growth. Based on the BLM co-incubation studies, we decided to repeat the studies on 1205Lu cells. So, 25 μM (at 34% cell growth) of vitamin-D3 and 50 μM (at 44% cell growth) of vitamin-A were used for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of cell growth (22%). Though co-incubation of vitamin-D3 with progesterone did not show any difference in cell growth (51%), yet co-incubation with RU-486 showed a decrease in 1205Lu cell growth (21%). Similarly co-incubation of vitamin-A with progesterone (25% cell growth) and with RU-486 (23% cell growth) showed a synergistic effect on the decrease of 1205Lu cell growth. Conclusion: These studies suggested that combination of vitamins and steroids might be effective in decreasing melanoma cell growth. Hence, various combination of vitamins and steroids will be tested for their effect on melanoma cell growth in vitro in order to develop a combo drug treatment for melanoma.

Antagonism of Growth Hormone Receptor Suppresses Cancer Growth and Drug Resistance in Mice

Abstract Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) play important roles in different stages of progression and drug resistance in many types of cancers, including breast, colon, endometrial, liver cancer and melanoma. GH receptor (GHR) is highly expressed in melanoma and promotes cancer proliferation and multidrug efflux pumps mediated drug resistance. Knockdown of GHR in melanoma cells significantly increased their drug sensitivity in vitro. Thus, a GHR antagonist could become a therapeutic molecule in suppressing melanoma cancer growth and sensitizing the tumor to chemotherapy in vivo. Here, we used GHR antagonist (GHA) transgenic mice which constitutively express a GHA to specifically suppress GH/IGF-1 axis. We found have circulating IGF-1 level was significantly lowered in these mice as a result of GHR antagonism. Furthermore, the sera from the mice could inhibit the growth of melanoma cells in culture. Recombinant GHA produced in our laboratory was able to suppress the phosphorylation of STAT5, a well-established marker of GH action, and the phosphorylation of MAPK, a critical signaling component of cell growth. The GHA mice were intradermally inoculated with mouse melanoma cells (B16-F10) or subcutaneously inoculated with mouse liver cancer cells (Hepa1-6) to generate syngeneic mouse tumor models. We observed that tumor size and tumor weight were markedly reduced and that phosphorylation of STAT5 and MAPK was suppressed in the livers from these mice. In parallel, the activation of GH signaling and the expression level of various types of multidrug efflux pumps were reduced in these tumors. To test the effect of GHA on drug synergy, the GHA mice or WT controls with liver cancer cells were treated with sorafenib or vehicle. Sorafenib is an FDA-approved tyrosine kinase inhibitor, widely used to treat advanced hepatocarcinoma, but has a reduced efficacy in application due to the multidrug efflux pump ABCG2. In vitro, recombinant bovine GH increased the IC50 of sorafenib and the expression of ABCG2. In vivo, GHA mice treated with sorafenib had the smallest tumors compared with WT mice, or in mice treated with sorafenib or GHA alone. Furthermore, ABCG2 mRNA levels were also suppressed in the liver tumors from GHA mice. All these findings from functional and mechanistic investigations confirm that a GHA or Pegvisomant is effective in cancer treatment in vivo and may be a novel therapeutic strategy or molecule to suppress the tumor growth and to sensitize different types of cancers to anti-cancer therapies. Acknowledgments: This work was supported by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll to J.K.; NIH-R01AG059779, the AMVETS, Edison Biotechnology Institute and Diabetes Institute at Ohio University; OURC funding and Baker Fund to Y.Q.; the PURF Fund and the John J. Kopchick Molecular Cell Biology Undergraduate Student Fund to N.A and J.T.

Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time.

Some secreted cysteine protease inhibitors of the cystatin family appear to affect intracellular proteolysis and growth of human cells, as a result of internalization. Here, we studied the effects of external addition of the most abundant human cystatin, cystatin C, on viability and proliferation of cancer cells in culture. A dose‐dependent decrease in viable cells was seen for A375 melanoma, MCF‐7 breast cancer, and PC‐3 prostate cancer cells cultured in 1–5 µm cystatin C after 24 h. Real‐time assessment of growth rates in A375 cell cultures for 48 h by digital holographic microscopy showed an increased doubling time for cells cultured in the presence of 5 µm cystatin C (20.1 h) compared with control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease in cell proliferation at the population level. Tracking of individual cells in phase holographic images showed that dividing cells incubated with 5 µm cystatin C underwent fewer mitoses during 48 h than control cells. In addition, the time between cell divisions was longer, especially for the first cell cycle. Incubation with the variant W106F‐cystatin C (with high cellular uptake rate) resulted in a lower number of viable cells and a prolonged doubling time than when cells were incubated with wild‐type cystatin C, but no effect was observed for (R24A,R25A)‐cystatin C (low cellular uptake). Thus, cystatin C causes prolonged cell division leading to decreased proliferation of melanoma cells, and internalization seems to be a prerequisite for this effect.

Combined Effect of Vitamin‐D3 and Progesterone on In‐Vitro Human Melanoma (BLM) Cell Growth

Our previous study showed that steroids especially progesterone and RU-486 significantly inhibited human melanoma (BLM) cell growth in-vitro. As lack of vitamin-D can lead to cancer and vitamin-D is synthesized in the skin by the UV rays, we decided to check the combined effect of vitamin-D3 and progesterone on human melanoma cell growth. Along with vitamin-D3, we also decided to include vitamin-A and RU-486.So our aimswere to check the in-vitro effects of active vitamin-D3 and vitamin-A combined with progesterone and RU-486 on human melanoma (BLM) cell growth. Initially dose-curve (100 nM to 100 µM concentration) study was carried out with vitamin-D3 and vitamin-A (retinoic acid) to determine the optimal concentrations of vitamins-D3 and A for co-incubation with progesterone (50 µM) and RU-486 (50 µM). Vitamin-D3 and vitamin-A showed a dose-dependent decrease in cell growth. Based on the dose-curve, it was decided to use 25 µM (at 57% cell growth) of vitamin-D3 and 50 µM (at 55% cell growth) of vitamin-A for co-incubation between them and also with progesterone and RU-486 separately. Co-incubation of vitamin-D3 and vitamin-A showed an additive effect on the decrease of cell growth (at 20%). Similarly co-incubation of vitamin-D3 with progesterone (at 33% cell growth) and RU-486 (at 28% cell growth) as well as co-incubation of vitamin-A with progesterone (at 31% cell growth) and RU-486 (at 18% cell growth) showed an additive effect on the decrease of cell growth. Since our previous study also showed the involvement of IL-8 in the regulation of melanoma cell growth, supernatants from the treated cells were subjected to Elisarray. There was a decrease in IL-8 secretion with vitamin-D3 treatment. But, vitamin-A alone treatment showed an increase in IL-8 secretion. However, co-incubation of vitamin-A with vitamin-D3, progesterone and RU-486 showed a decrease in IL-8 secretion. These studies suggested that a combination of vitamins and steroids might be effective in decreasing melanoma cell growth than vitamin or steroid alone. Conclusion Co-incubation of vitamins with steroids indicated a combo drug effect on the decrease of melanoma cell growth. Hence, various combinations of vitamin-D3, vitamin-A and steroids would be tested for their effect on melanoma cell growth in vitro.

ITIH5, a p53-responsive gene, inhibits the growth and metastasis of melanoma cells by downregulating the transcriptional activity of KLF4

ITIH5, a member of the inter-α-trypsin inhibitory (ITI) gene family, acts as a putative tumour-suppressor gene in many cancers. However, its role and the regulatory mechanism in melanoma are still unclear. Here, we found that the expression of ITIH5 was decreased in melanoma tissues compared with normal skin tissues. Decreased expression of ITIH5 was correlated with clinicopathological features and predicted poor prognosis in patients with melanoma. Forced expression of ITIH5 significantly inhibited melanoma cell proliferation and metastasis in vitro and ex vivo while knockdown of ITIH5 expression enhanced the malignant behaviour of melanoma cells. In further mechanistic studies, we showed that p53 can directly bind to the promoter of ITIH5 and thus promotes transcription of ITIH5 in melanoma cells. Additionally, we found that ITIH5 interacted with Krüppel-like factor 4 (KLF4) and inhibited its transcriptional activity. Collectively, our data not only identified a tumour-suppressive role of ITIH5 in melanoma but also revealed that upregulation of ITIH5 by p53 suppressed melanoma cell growth and migration likely by downmodulating the transcriptional activity of KLF4.

USP8 is a Novel Therapeutic Target in Melanoma Through Regulating Receptor Tyrosine Kinase Levels.

IntroductionThe hyperactivation of receptor tyrosine kinase (RTK)-mediated pathways plays an important role in melanoma progression and resistance to therapy. The ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme and its inhibition induces degradation of RTKs. This work explored the expression and role of USP8 in melanoma.MethodsELISA and qPCR were performed to assess USP8 expression in melanoma tissues and cells, as well as their normal counterparts. Cellular proliferation, migration and apoptosis assays were performed to determine USP8 functions in three melanoma cell lines. Western blot was performed to analyze RTK signaling in melanoma cells after USP8 inhibition.ResultsmRNA and protein level of USP8 were higher in melanoma cells than normal melanocytes. Higher USP8 expression was also found in tumors in the majority of melanoma patients. USP8 expression was not associated with clinicopathological features, such as age, disease stage, histology, ulceration and BRAF status. Functional analysis demonstrated that USP8 overexpression promoted melanoma cell activities and alleviated the inhibitory effects of therapeutic drugs. In contrast, USP8 knockdown suppressed melanoma cell growth, survival and migration, and augmented the inhibitory effects of therapeutic drugs. Mechanism studies revealed that USP8 inhibition remarkably reduced the expression level of multiple oncogenic RTKs, including c-Met, Kit, EGFR and GPCR. Consistently, RTK-mediated downstream pathways were disrupted in USP8-depleted cells, leading to the increased level of pro-apoptotic proteins and decreased level of anti-apoptotic proteins.ConclusionInhibition of USP8 activity is a novel sensitizing strategy to overcome therapy resistance in melanoma.

Control of Breast Cancer Pathogenesis by Histone Methylation and the Hairless Histone Demethylase.

Breast cancer is a highly heterogeneous disease, encompassing many subtypes that have distinct origins, behaviors, and prognoses. Although traditionally seen as a genetic disease, breast cancer is now also known to involve epigenetic abnormalities. Epigenetic regulators, such as DNA methyltransferases and histone-modifying enzymes, play essential roles in gene regulation and cancer development. Dysregulation of epigenetic regulator activity has been causally linked with breast cancer pathogenesis. Hairless (HR) encodes a 130-kDa transcription factor that is essential for development and tissue homeostasis. Its role in transcription regulation is partly mediated by its interaction with multiple nuclear receptors, including thyroid hormone receptor, retinoic acid receptor-related orphan receptors, and vitamin D receptor. HR has been studied primarily in epidermal development and homeostasis. Hr-mutant mice are highly susceptible to ultraviolet- or carcinogen-induced skin tumors. Besides its putative tumor suppressor function in skin, loss of HR function has also been implicated in increased leukemia susceptibility and promotes the growth of melanoma and brain cancer cells. HR has also been demonstrated to function as a histone H3 lysine 9 demethylase. Recent genomics studies have identified HR mutations in a variety of human cancers, including breast cancer. The anticancer function and mechanism of action by HR in mammary tissue remains to be investigated. Here, we review the emerging role of HR, its histone demethylase activity and histone methylation in breast cancer development, and potential for epigenetic therapy.

Fibroblast MMP14-Dependent Collagen Processing Is Necessary for Melanoma Growth.

Simple SummaryMatrix metalloproteinases (MMPs) were considered as targets for the treatment of various cancers. However, initial trials using broad inhibitors to MMPs have failed, partly attributed to the contrasting functions of these proteases acting as tumor promoters and suppressors, among other reasons. Our data now suggest that specific inhibition of MMP14 might represent a more specific approach, as loss of this protease in fibroblasts resulted in reduced growth of grafted melanomas. Here, we found that deletion of MMP14 in fibroblasts generates a matrix-rich environment that reduces tumor vascularization and melanoma cell proliferation. In in vitro and ex vivo assays, we showed that the latter is mediated by stiffening of the tissue due to collagen accumulation. Additionally, in vivo, we show that independently of MMP14 deletion, a collagen-rich stiff matrix inhibits the growth of melanomas.Skin homeostasis results from balanced synthesis and degradation of the extracellular matrix in the dermis. Deletion of the proteolytic enzyme MMP14 in dermal fibroblasts (MMP14Sf−/−) leads to a fibrotic skin phenotype with the accumulation of collagen type I, resulting from impaired proteolysis. Here, we show that melanoma growth in these mouse fibrotic dermal samples was decreased, paralleled by reduced tumor cell proliferation and vessel density. Using atomic force microscopy, we found increased peritumoral matrix stiffness of early but not late melanomas in the absence of fibroblast-derived MMP14. However, total collagen levels were increased at late melanoma stages in MMP14Sf−/− mice compared to controls. In ex vivo invasion assays, melanoma cells formed smaller tumor islands in MMP14Sf−/− skin, indicating that MMP14-dependent matrix accumulation regulates tumor growth. In line with these data, in vitro melanoma cell growth was inhibited in high collagen 3D spheroids or stiff substrates. Most importantly, in vivo induction of fibrosis using bleomycin reduced melanoma tumor growth. In summary, we show that MMP14 expression in stromal fibroblasts regulates melanoma tumor progression by modifying the peritumoral matrix and point to collagen accumulation as a negative regulator of melanoma.

Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance.

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394.

Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance.

Simple SummaryMelanoma is a highly metastatic and therapy-resistant cancer and is therefore associated with low survival rates of patients. In melanoma, the inactivation of the wild-type form of the p53 tumour suppressor protein is a frequent event, mainly through interactions with MDM2 and MDMX. In this work, our recently disclosed p53-activating agent, SLMP53-2, displayed promising in vitro and in vivo antitumour activity, with particular impacts on melanoma migration and invasion. Moreover, SLMP53-2 (re)sensitized melanoma cells to clinically used chemotherapeutic agents, potentially overcoming the therapeutic resistance issue. As a whole, the p53 activator SLMP53-2 may represent a new therapeutic opportunity for melanoma, particularly in combination with MAPK pathway-targeting drugs.Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53–MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.