T-Type Calcium Channels: A Potential Novel Target in Melanoma

T-type calcium channels (TTCCs) are overexpressed in several cancers. In this review, we summarize the recent advances and new insights into TTCC biology, tumor progression, and prognosis biomarker and therapeutic potential in the melanoma field. We describe a novel correlation between the Cav3.1 isoform and the increased basal autophagy in BRAFV600E-mutant melanomas and after acquired resistance to BRAF inhibitors. Indeed, TTCC blockers reduce melanoma cell viability and migration/invasion in vitro and tumor growth in mice xenografts in both BRAF-inhibitor-sensitive and -resistant scenarios. These studies open a new, promising therapeutic approach for disseminated melanoma and improved treatment in BRAFi relapsed melanomas, but further validation and clinical trials are needed for it to become a real therapeutic optionThis work was supported by grants from ISCIII/FEDER “Una manera de hacer Europa” (PI1500711 to RMM; PI18/00573 to RMM and AM). CB and PS hold a pre-doctoral fellowship from UdL-IRBLleida. RI holds a pre-doctoral fellowship from Asociación Española Contra el Cancer (AECC), Catalunya Contra el Cancer, Lleida. AM holds a post-doctoral fellowship from Asociación Española Contra el Cancer (AECC)

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine

An inducible knockout mouse to model the cellautonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifeninducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors

Downregulation of epidermal growth factor receptor in hepatocellular carcinoma facilitates transforming growth factor-β-induced epithelial to amoeboid transition

The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-β) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-β is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-β is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-β-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-β upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFB1 or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-β-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-β pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic

Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues

In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01).The study was done according to the research collaboration with Dako Denmark A/S. The research team was also supported by grants FIS PI100922, Fundación Mutua Madrileña AP75732010, 2009SGR794, RD12/0036/0013, Fundación Asociación Española contra el Cancer, programa de intensificación de la investigación, Instituto Carlos III, Verelst Baarmoederkankerfonds, Leuven, and European Network for Individualized Treatment of Endometrial Carcinoma. F. A. is senior researcher for the research fund Flandersb. Tumor samples were obtained with the support of XarxaCatalana de Bancs de Tumors, the TumorBanc Platform of RTICC, and Red de Biobancos (RD09/0076/00059

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics

Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.This work was supported by grants from ISCIII and FEDER (“una manera de hacerEuropa”) PI15/00711 to RMM and PI18/00573 to RMM & AM. CIBERONC (CB16/12/00231) to XMG.Postdoctoral AECC (POSTD004MACI - POSTD16) to AM. Predoctoral UdL to PS and predoctoralAECC fellowship to IR. The work was also supported by the Xarxa de Bancs de Tumors de Catalunyasponsored by “Pla Director d’Oncologia de Catalunya (XBTC)”and IRBLleida Biobank (B.0000682)and PLATAFORMA BIOBANCOS (PT17/0015/0027; PT20/00021

FISH analysis of PTEN in endometrial carcinoma. Comparison with SNP arrays and MLPA

Aims: To check the usefulness of a standardized protocol of PTEN FISH in 31 endometrial carcinomas (ECs) in comparison with SNP array (SNPA), multiplex ligation-dependent probe amplification (MLPA), and immunohistochemistry. Methods and results: Fluorescence in-situ hybridization analysis showed two PTEN copies in 17 cases, three copies in nine cases, hemizygous deletion in two cases, and diverse cell populations with different PTEN copy number in three cases. A good correlation was seen between FISH and SNPA, particularly in cases with three copies. FISH identified two cases with entire deletion of chromosome 10, but did not identify a focal deletion of PTEN. Five cases with PTEN deletion and duplication of the second allele by SNPA were interpreted as normal by FISH. Concordance between FISH and MLPA was seen in 15 cases with two copies, and in two cases with PTEN deletion. Six cases were interpreted as amplified by MLPA, but showed polyploidy by FISH. FISH was superior to SNPA and MLPA in assessing the tumours with diverse cell populations with different PTEN copies. Conclusions: The results show good concordance between FISH, SNPA and MLPA. SNPA was superior in tumours with deletion of one copy and duplication of the second allele. FISH was superior in assessing tumour heterogeneity.The study was supported by a research agreement with Dako, Denmark. The research team was also supported by grants FIS PI100922, Fundacion Mutua Madrilena AP75732010, 2009SGR794, RD12/0036/ ~ 0013, Fundacion Asociaci on Espa nola contra el Can- ~ cer, Programa de Intensificacion de la Investigaci on, Instituto Carlos III, Verelst Baarmoederkankerfonds, Leuven, and ENITEC (European Network for Individualized Treatment of Endometrial Carcinoma). F. Amant is senior researcher for the research fund Flandersb (FWO). Tumour samples were obtained with the support of Xarxa Catalana de Bancs de Tumours, and Plataforma de Biobancos ISCIII (PT13/ 0010/0014)

WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy. Amoeboid cells are associated with melanoma invasive capacity. Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours