TIMP-2 secreted by monocyte-like cells is a potent suppressor of invadopodia formation in pancreatic cancer cells.

BACKGROUND: Monocytes are a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC). However, the complex interactions between tumor cells and monocytes and their role in tumor invasion have not been fully established. METHODS: To specifically test the impact of interaction on invasive potential two PDAC cell lines PaTu8902 and CFPAC-1 were selected on their ability to form invasive adhesions, otherwise known as invadopodia and invade in a spheroid invasion assay. RESULTS: Interestingly when the PDAC cells were co-cultured with undifferentiated THP1 monocyte-like cells invadopodia formation was significantly suppressed. Moreover, conditioned media of THP1 cells (CM) was also able to suppress invadopodia formation. Further investigation revealed that both tissue inhibitor of metalloproteinase (TIMP) 1 and 2 were present in the CM. However, suppression of invadopodia formation was found that was specific to TIMP2 activity. CONCLUSIONS: Our findings indicate that TIMP2 levels in the tumour microenvironment may have prognostic value in patients with PDAC. Furthermore, activation of TIMP2 expressing monocytes in the primary tumour could present a potential therapeutic opportunity to suppress cell invasion in PDAC.

High Throughput Fluorescence-Based In Vitro Experimental Platform for the Identification of Effective Therapies to Overcome Tumour Microenvironment-Mediated Drug Resistance in AML.

The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment.

SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML.

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3ß, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia.

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression.

Originally considered to act as a transcriptional co-factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient's clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow-cycling JARID1B gene.

Terfenadine induces apoptosis and autophagy in melanoma cells through ROS-dependent and -independent mechanisms.

Previously we found that terfenadine, an H1 histamine receptor antagonist, acts as a potent apoptosis inducer in melanoma cells through modulation of Ca(2+) homeostasis. In this report, focusing our attention on the apoptotic mechanisms activated by terfenadine, we show that this drug can potentially activate distinct intrinsic signaling pathways depending on culture conditions. Serum-deprived conditions enhance the cytotoxic effect of terfenadine and caspase-4 and -2 are activated upstream of caspase-9. Moreover, although we found an increase in ROS levels, the apoptosis was ROS independent. Conversely, terfenadine treatment in complete medium induced ROS-dependent apoptosis. Caspase-4, -2, and -9 were simultaneously activated and p73 and Noxa induction were involved. ROS inhibition prevented p73 and Noxa expression but not p53 and p21 expression, suggesting a role for Noxa in p53-independent apoptosis in melanoma cells. Finally, we found that terfenadine induced autophagy, that can promote apoptosis. These findings demonstrate the great potential of terfenadine to kill melanoma cells through different cellular signaling pathways and could contribute to define new therapeutic strategies in melanoma.

The diversity profile of TP53 is influenced by positive selection on the immediately upstream locus WDR79.

BACKGROUND/AIM: TP53 is an efficient central node in a signal transduction network that responds to minimize cancer. However, over 50% of tumors show some mutation in TP53. Thus, one might argue that this single central node network lacks robustness. Therefore, we wanted to investigate if natural selection has played a role in shaping the genomic region containing TP53. METHODS: We have analyzed the HapMap data for evidence of selection using F(ST) pairwise comparisons and the extended haplotype homozygosity test on a 200-kb region encompassing TP53. We have also resequenced 4 kb upstream TP53 in Europeans (including melanoma patients), Asians, Australian Aborigines and Africans. RESULTS: Genetic hitchhiking by a linked, positively selected allele at the nearby gene WDR79 may be partly responsible for the sequence diversity profile of TP53. It can help explain why the TP53 Arg72 allele is the major allele in Europeans even when the alternative allele, 72Pro, has been reported to offer an increased longevity after disease. CONCLUSIONS: Despite the important role of TP53, a complex interplay with other evolutionary forces, which are extrinsic to TP53 function, may have driven the genetic diversity pattern of this locus, and, as a consequence, its structure and function.

RKIP Regulates Differentiation-Related Features in Melanocytic Cells.

Raf Kinase Inhibitor Protein (RKIP) has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancers. However, little is known about RKIP in melanoma or regarding its function in normal cells. We examined the role of RKIP in both primary melanocytes and malignant melanoma cells and evaluated its diagnostic and prognostic value. IHC analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. As a whole, our data support the utility of RKIP as a diagnostic marker for early-stage melanomas. In addition, these findings indicate its participation in the maintenance of a differentiated state of melanocytic cells by modulating genes intimately linked to the cellular motility and explain the progressive decrease of RKIP often described in tumors.

Terfenadine-induced apoptosis in human melanoma cells is mediated through Ca2+ homeostasis modulation and tyrosine kinase activity, independently of H1 histamine receptors.

In our previous works, we have demonstrated that terfenadine (TEF) induces DNA damage and apoptosis in human melanoma cell lines. In this present work, we have studied the effect of histamine on viability of A375 human melanoma cells and the cell-signalling pathways through which TEF may induce its apoptotic effect. We have found that exogenous histamine stimulates A375 melanoma cell proliferation in a dose- and time-dependent manner. Moreover, TEF-induced apoptosis seems to occur via other cellular pathways independent of the histamine-signalling system since co-treatment of histamine with TEF did not protect melanoma cells from the cytotoxic effect of TEF, and alpha fluoromethylhistidine did not induce the same cytotoxic effect of TEF. In addition, we have observed that knocking down the H1 histamine receptor (HRH1) by small interference RNA approach protects melanoma cells only slightly from TEF-induced apoptosis. To explore the molecular mechanisms responsible for histamine and TEF effect on the cell growth, we analysed intracellular cyclic nucleotides and Ca(2+) levels. TEF did not modify intracellular levels of cyclic adenosine 3',5'-monophosphate and cyclic guanine 3',5'-monophosphate; however, TEF induced a very sharp and sustained increase in cytosolic Ca(2+) levels in A375 melanoma cells. On the contrary, histamine did not modulate intracellular Ca(2+). TEF-induced Ca(2+) rise and apoptosis appear to be phospholipase C (PLC) dependent since neomycin and U73122, two inhibitors of PLC, abolished cytosolic Ca(2+) increase and protected the cells completely from cell death. Furthermore, inhibition of tyrosine kinase activity by genistein blocked cytosolic Ca(2+) rise and TEF-induced apoptosis. These results suggest that TEF modulates Ca(2+) homeostasis and induces apoptosis through other cellular pathways involving tyrosine kinase activity, independently of HRH1.

Involvement of ANXA5 and ILKAP in susceptibility to malignant melanoma.

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12-1.48; p-value = 4×10-4). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes.

A customized pigmentation SNP array identifies a novel SNP associated with melanoma predisposition in the SLC45A2 gene.

As the incidence of Malignant Melanoma (MM) reflects an interaction between skin colour and UV exposure, variations in genes implicated in pigmentation and tanning response to UV may be associated with susceptibility to MM. In this study, 363 SNPs in 65 gene regions belonging to the pigmentation pathway have been successfully genotyped using a SNP array. Five hundred and ninety MM cases and 507 controls were analyzed in a discovery phase I. Ten candidate SNPs based on a p-value threshold of 0.01 were identified. Two of them, rs35414 (SLC45A2) and rs2069398 (SILV/CKD2), were statistically significant after conservative Bonferroni correction. The best six SNPs were further tested in an independent Spanish series (624 MM cases and 789 controls). A novel SNP located on the SLC45A2 gene (rs35414) was found to be significantly associated with melanoma in both phase I and phase II (P<0.0001). None of the other five SNPs were replicated in this second phase of the study. However, three SNPs in TYR, SILV/CDK2 and ADAMTS20 genes (rs17793678, rs2069398 and rs1510521 respectively) had an overall p-value<0.05 when considering the whole DNA collection (1214 MM cases and 1296 controls). Both the SLC45A2 and the SILV/CDK2 variants behave as protective alleles, while the TYR and ADAMTS20 variants seem to function as risk alleles. Cumulative effects were detected when these four variants were considered together. Furthermore, individuals carrying two or more mutations in MC1R, a well-known low penetrance melanoma-predisposing gene, had a decreased MM risk if concurrently bearing the SLC45A2 protective variant. To our knowledge, this is the largest study on Spanish sporadic MM cases to date.