Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

BACKGROUND: Gallbladder cancer (GBC) is a prevalent and deadly biliary tract carcinoma, often diagnosed at advanced stages with limited treatment options. The 5-year survival rate varies widely from 4 to 60%, mainly due to differences in disease stage detection. With only a small fraction of patients having resectable tumors and a high incidence of metastasis, advanced GBC stages are characterized by significant chemoresistance. Identification of new therapeutic targets is crucial, and recent studies have shown that the Endothelin-1 (ET-1) signaling pathway, involving ETAR and/or ETBR receptors (ETRs), plays a crucial role in promoting tumor aggressiveness in various cancer models. Blocking one or both receptors has been reported to reduce invasiveness and chemoresistance in cancers like ovarian, prostate, and colon. Furthermore, transcriptomic studies have associated ET-1 levels with late stages of GBC; however, it remains unclear whether its signaling or its inhibition has implications for its aggressiveness. Although the role of ET-1 signaling in gallbladder physiology is minimally understood, its significance in other tumor models leads us to hypothesize its involvement in GBC malignancy. RESULTS: In this study, we investigated the expression of ET-1 pathway proteins in three GBC cell lines and a primary GBC culture. Our findings demonstrated that both ETAR and ETBR receptors are expressed in GBC cells and tumor samples. Moreover, we successfully down-regulated ET-1 signaling using a non-selective ETR antagonist, Macitentan, which resulted in reduced migratory and invasive capacities of GBC cells. Additionally, Macitentan treatment chemosensitized the cells to Gemcitabine, a commonly used therapy for GBC. CONCLUSION: For the first time, we reveal the role of the ET-1 pathway in GBC cells, providing insight into the potential therapeutic targeting of its receptors to mitigate invasion and chemoresistance in this cancer with limited treatment options. These findings pave the way for further exploration of Macitentan or other ETR antagonists as potential therapeutic strategies for GBC management. In summary, our study represents a groundbreaking contribution to the field by providing the first evidence of the ET 1 pathway's pivotal role in modulating the behavior and aggressiveness of GBC cells, shedding new light on potential therapeutic targets.

Association of Germline Variation in Driver Genes with Breast Cancer Risk in Chilean Population.

Cancer is a genomic disease, with driver mutations contributing to tumorigenesis. These potentially heritable variants influence risk and underlie familial breast cancer (BC). This study evaluated associations between BC risk and 13 SNPs in driver genes MAP3K1, SF3B1, SMAD4, ARID2, ATR, KMT2C, MAP3K13, NCOR1, and TBX3, in BRCA1/2-negative Chilean families. SNPs were genotyped using TaqMan Assay in 492 cases and 1285 controls. There were no associations between rs75704921:C>T (ARID2); rs2229032:A>C (ATR); rs3735156:C>G (KMT2C); rs2276738:G>C, rs2293906:C>T, rs4075943T:>A, rs13091808:C>T (MAP3K13); rs178831:G>A (NCOR1); or rs3759173:C>A (TBX3) and risk. The MAP3K1 rs832583 A allele (C/A+A/A) showed a protective effect in families with moderate BC history (OR = 0.7 [95% CI 0.5-0.9] p = 0.01). SF3B1 rs16865677-T (G/T+T/T) increased risk in sporadic early-onset BC (OR = 1.4 [95% CI 1.0-2.0] p = 0.01). SMAD4 rs3819122-C (A/C+C/C) increased risk in cases with moderate family history (OR = 2.0 [95% CI 1.3-2.9] p ≤ 0.0001) and sporadic cases diagnosed ≤50 years (OR = 1.6 [95% CI 1.1-2.2] p = 0.006). SMAD4 rs12456284:A>G increased BC risk in G-allele carriers (A/G + G/G) in cases with ≥2 BC/OC cases and early-onset cases (OR = 1.2 [95% CI 1.0-1.6] p = 0.04 and OR = 1.4 [95% CI 1.0-1.9] p = 0.03, respectively). Our study suggests that specific germline variants in driver genes MAP3K1, SF3B1, and SMAD4 contribute to BC risk in Chilean population.

Indomethacin Induces Spermidine/Spermine-N1-Acetyltransferase-1 via the Nucleolin-CDK1 Axis and Synergizes with the Polyamine Oxidase Inhibitor Methoctramine in Lung Cancer Cells.

Indomethacin is a non-selective NSAID used against pain and inflammation. Although cyclooxygenase (COX) inhibition is considered indomethacin's primary action mechanism, COX-independent ways are associated with beneficial effects in cancer. In colon cancer cells, the activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) is related to the increase in spermidine/spermine-N1-acetyltransferase-1 (SSAT-1), a key enzyme for polyamine degradation, and related to cell cycle arrest. Indomethacin increases the SSAT-1 levels in lung cancer cells; however, the mechanism relying on the SSAT-1 increase is unclear. Thus, we asked for the influence of the PPAR-γ on the SSAT-1 expression in two lung cancer cell lines: H1299 and A549. We found that the inhibition of PPAR-γ with GW9662 did not revert the increase in SSAT-1 induced by indomethacin. Because the mRNA of SSAT-1 suffers a pre-translation retention step by nucleolin, a nucleolar protein, we explored the relationship between indomethacin and the upstream translation regulators of SSAT-1. We found that indomethacin decreases the nucleolin levels and the cyclin-dependent kinase 1 (CDK1) levels, which phosphorylates nucleolin in mitosis. Overexpression of nucleolin partially reverts the effect of indomethacin over cell viability and SSAT-1 levels. On the other hand, Casein Kinase, known for phosphorylating nucleolin during interphase, is not modified by indomethacin. SSAT-1 exerts its antiproliferative effect by acetylating polyamines, a process reverted by the polyamine oxidase (PAOX). Recently, methoctramine was described as the most specific inhibitor of PAOX. Thus, we asked if methoctramine could increase the effect of indomethacin. We found that, when combined, indomethacin and methoctramine have a synergistic effect against NSCLC cells in vitro. These results suggest that indomethacin increases the SSAT-1 levels by reducing the CDK1-nucleolin regulatory axis, and the PAOX inhibition with methoctramine could improve the antiproliferative effect of indomethacin.

Association of FANCM Mutations with Familial and Early-Onset Breast Cancer Risk in a South American Population.

Breast cancer (BC) is the most common cancer among women worldwide. BRCA1/2 are responsible for 16-20% of the risk for hereditary BC. Other susceptibility genes have been identified; Fanconi Anemia Complementation Group M (FANCM) being one of these. Two variants in FANCM, rs144567652 and rs147021911, are associated with BC risk. These variants have been described in Finland, Italy, France, Spain, Germany, Australia, the United States, Sweden, Finnish, and the Netherlands, but not in the South American populations. Our study evaluated the association of the SNPs rs144567652 and rs147021911 with BC risk in non-carriers of BRCA1/2 mutations from a South American population. The SNPs were genotyped in 492 BRCA1/2-negative BC cases and 673 controls. Our data do not support an association between FANCM rs147021911 and rs144567652 SNPs and BC risk. Nevertheless, two BC cases, one with a family history of BC and the other with sporadic early-onset BC, were C/T heterozygotes for rs144567652. In conclusion, this is the first study related contribution of FANCM mutations and BC risk in a South American population. Nevertheless, more studies are necessary to evaluate if rs144567652 could be responsible for familial BC in BRCA1/2-negatives and for early-onset non-familial BC in Chilean BC cases.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

A Single Variant in Pri-miRNA-155 Associated with Susceptibility to Hereditary Breast Cancer Promotes Aggressiveness in Breast Cancer Cells.

Variants in genes encoding for microRNAs have been associated with their deregulation in breast cancer (BC). Sequencing of microRNAs deregulated in BC was performed using DNA from Chilean patients with a strong family history and negative for mutations in BRCA1/BRCA2. Seventeen variants were identified, three of which were selected for a case-control association study: rs376491654 (miR-335), rs755634302 (miR-497), and rs190708267 (miR-155). For rs190708267 C>T, the heterozygous T allele was detected in four BC cases and absent in controls, while homozygous TT cases were not detected. Variants were modelled in silico, cloned in a plasmid, expressed in BC cell lines, and functional in vitro assays were performed. Overexpression of the miR-155-T allele increased mature miR-155-5p levels in both BC cell lines, suggesting that its presence alters pre-miR-155 processing. Moreover, BC cells overexpressing the miR-155-T allele showed increased proliferation, migration, and resistance to cisplatin-induced death compared to miR-155-C overexpressing cells. Of note, the 3'UTR of APC, GSK3ß, and PPP1CA genes, all into the canonical Wnt signaling pathway, were identified as direct targets. APC and GSK3ß mRNA levels decreased while PP1 levels increased. These results suggest a pathogenic role of the variant rs190708267 (miR-155) in BRCA 1/2 negative BC, conferring susceptibility and promoting traits of aggressiveness.

Characterization of High-Risk HPV/EBV Co-Presence in Pre-Malignant Cervical Lesions and Squamous Cell Carcinomas.

High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. However, a low proportion of HR-HPV-infected women finally develop this cancer, which suggests the involvement of additional cofactors. Epstein−Barr virus (EBV) has been detected in cervical squamous cell carcinomas (SCCs) as well as in low- (LSIL) and high-grade (HSIL) squamous intraepithelial lesions, although its role is unknown. In this study, we characterized HR-HPV/EBV co-presence and viral gene expression in LSIL (n = 22), HSIL (n = 52), and SCC (n = 19) from Chilean women. Additionally, phenotypic changes were evaluated in cervical cancer cells ectopically expressing BamHI-A Rightward Frame 1 (BARF1). BARF1 is a lytic gene also expressed in EBV-positive epithelial tumors during the EBV latency program. HPV was detected in 6/22 (27.3%) LSIL, 38/52 (73.1%) HSIL, and 15/19 (78.9%) SCC cases (p < 0.001). On the other hand, EBV was detected in 16/22 (72.7%) LSIL, 27/52 (51.9%) HSIL, and 13/19 (68.4%) SCC cases (p = 0.177). HR-HPV/EBV co-presence was detected in 3/22 (13.6%) LSIL, 17/52 (32.7%) HSIL, and 11/19 (57.9%) SCC cases (p = 0.020). Additionally, BARF1 transcripts were detected in 37/55 (67.3%) of EBV positive cases and in 19/30 (63.3%) of HR-HPV/EBV positive cases. Increased proliferation, migration, and epithelial-mesenchymal transition (EMT) was observed in cervical cancer cells expressing BARF1. Thus, both EBV and BARF1 transcripts are detected in low- and high-grade cervical lesions as well as in cervical carcinomas. In addition, BARF1 can modulate the tumor behavior in cervical cancer cells, suggesting a role in increasing tumor aggressiveness.

Heritable genomic diversity in breast cancer driver genes and associations with risk in a Chilean population.

BACKGROUND: Driver mutations are the genetic components responsible for tumor initiation and progression. These variants, which may be inherited, influence cancer risk and therefore underlie many familial cancers. The present study examines the potential association between SNPs in driver genes SF3B1 (rs4685), TBX3 (rs12366395, rs8853, and rs1061651) and MAP3K1 (rs72758040) and BC in BRCA1/2-negative Chilean families. METHODS: The SNPs were genotyped in 486 BC cases and 1258 controls by TaqMan Assay. RESULTS: Our data do not support an association between rs4685:C > T, rs8853:T > C, or rs1061651:T > C and BC risk. However, the rs12366395-G allele (A/G + G/G) was associated with risk in families with a strong history of BC (OR = 1.2 [95% CI 1.0-1.6] p = 0.02 and OR = 1.5 [95% CI 1.0-2.2] p = 0.02, respectively). Moreover, rs72758040-C was associated with increased risk in cases with a moderate-to-strong family history of BC (OR = 1.3 [95% CI 1.0-1.7] p = 0.02 and OR = 1.3 [95% CI 1.0-1.8] p = 0.03 respectively). Finally, risk was significantly higher in homozygous C/C cases from families with a moderate-to-strong BC history (OR = 1.8 [95% CI 1.0-3.1] p = 0.03 and OR = 1.9 [95% CI 1.1-3.4] p = 0.01, respectively). We also evaluated the combined impact of rs12366395-G and rs72758040-C. Familial BC risk increased in a dose-dependent manner with risk allele count, reflecting an additive effect (p-trend = 0.0002). CONCLUSIONS: Our study suggests that germline variants in driver genes TBX3 (rs12366395) and MAP3K1 (rs72758040) may influence BC risk in BRCA1/2-negative Chilean families. Moreover, the presence of rs12366395-G and rs72758040-C could increase BC risk in a Chilean population.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/ß-catenin pathway and activity of nuclear ß-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3ß, while increasing active ß-catenin. It also increased the activity of nuclear ß-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the ß-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

Heritable genomic diversity in breast cancer driver genes and associations with risk in a Chilean population

BACKGROUND: Driver mutations are the genetic components responsible for tumor initiation and progression. These variants, which may be inherited, influence cancer risk and therefore underlie many familial cancers. The present study examines the potential association between SNPs in driver genes SF3B1 (rs4685), TBX3 (rs12366395, rs8853, and rs1061651) and MAP3K1 (rs72758040) and BC in BRCA1/2-negative Chilean families. METHODS: The SNPs were genotyped in 486 BC cases and 1258 controls by TaqMan Assay. RESULTS: Our data do not support an association between rs4685:C > T, rs8853:T > C, or rs1061651:T > C and BC risk. However, the rs12366395-G allele (A/G + G/G) was associated with risk in families with a strong history of BC (OR = 1.2 [95% CI 1.0-1.6] p = 0.02 and OR = 1.5 [95% CI 1.0-2.2] p = 0.02, respectively). Moreover, rs72758040-C was associated with increased risk in cases with a moderate-to-strong family history of BC (OR = 1.3 [95% CI 1.0-1.7] p = 0.02 and OR = 1.3 [95% CI 1.0-1.8] p = 0.03 respectively). Finally, risk was significantly higher in homozygous C/C cases from families with a moderate-to-strong BC history (OR = 1.8 [95% CI 1.0-3.1] p = 0.03 and OR = 1.9 [95% CI 1.1-3.4] p = 0.01, respectively). We also evaluated the combined impact of rs12366395-G and rs72758040-C. Familial BC risk increased in a dose-dependent manner with risk allele count, reflecting an additive effect (p-trend = 0.0002). CONCLUSIONS: Our study suggests that germline variants in driver genes TBX3 (rs12366395) and MAP3K1 (rs72758040) may influence BC risk in BRCA1/2-negative Chilean families. Moreover, the presence of rs12366395-G and rs72758040-C could increase BC risk in a Chilean population.

Genetic Variation in MicroRNA-423 Promotes Proliferation, Migration, Invasion, and Chemoresistance in Breast Cancer Cells.

MicroRNA-423 (miR-423) is highly expressed in breast cancer (BC). Previously, our group showed that the SNP rs6505162:C>A located in the pre-miR-423 was significantly associated with increased familial BC risk in patients with a strong family history of BC. Therefore, in this study, we evaluated the functional role of rs6505162 in mammary tumorigenesis in vitro to corroborate the association of this SNP with BC risk. We found that rs6505162:C>A upregulated expression of both mature miR-423 sequences (3p and 5p). Moreover, pre-miR-423-A enhanced proliferation, and promoted cisplatin resistance in BC cell lines. We also showed that pre-miR-423-A expression decreased cisplatin-induced apoptosis, and increased BC cell migration and invasion. We propose that the rs6505162-A allele promotes miR-423 overexpression, and that the rs6505162-A allele induces BC cell proliferation, viability, chemoresistance, migration, and invasion, and decreases cell apoptosis as a consequence. We suggest that rs6505162:C>A is a functional SNP site with potential utility as a marker for early diagnosis, prognosis, and treatment efficacy monitoring in BRCA1/2-negative BC patients, as well as a possible therapeutic target.

Phosphorylation of Endothelin-Converting Enzyme-1c at Serines 18 and 20 by CK2 Promotes Aggressiveness Traits in Colorectal Cancer Cells.

Endothelin-converting enzyme-1 (ECE1) activates the endothelin-1 peptide, which upregulates pathways that are related to diverse hallmarks of cancer. ECE1 is expressed as four isoforms differing in their N-terminal domains. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE1c, enhancing its stability and promoting invasiveness of colorectal cancer cells. However, the specific residues in ECE1c that are phosphorylated by CK2 and how this phosphorylation promotes invasiveness was unknown. Here we demonstrate that Ser-18 and Ser-20 are the bona fide residues phosphorylated by CK2 in ECE1c. Thus, biphospho-mimetic ECE1cDD and biphospho-resistant ECE1cAA mutants were constructed and stably expressed in different colorectal cancer cells through lentiviral transduction. Biphospho-mimetic ECE1cDD displayed the highest stability in cells, even in the presence of the specific CK2 inhibitor silmitasertib. Concordantly, ECE1cDD-expressing cells showed enhanced hallmarks of cancer, such as proliferation, migration, invasiveness, and self-renewal capacities. Conversely, cells expressing the less-stable biphospho-resistant ECE1cAA showed a reduction in these features, but also displayed an important sensitization to 5-fluorouracil, an antineoplastic agent traditionally used as therapy in colorectal cancer patients. Altogether, these findings suggest that phosphorylation of ECE1c at Ser-18 and Ser-20 by CK2 promotes aggressiveness in colorectal cancer cells. Therefore, phospho-ECE1c may constitute a novel biomarker of poor prognosis and CK2 inhibition may be envisioned as a potential therapy for colorectal cancer patients.

Human Papillomavirus 16 E7 Promotes EGFR/PI3K/AKT1/NRF2 Signaling Pathway Contributing to PIR/NF-κB Activation in Oral Cancer Cells.

A subset of oral carcinomas is etiologically related to high-risk human papillomavirus (HR-HPV) infection, with HPV16 being the most frequent HR-HPV type found in these carcinomas. The oncogenic role of HR-HPV is strongly dependent on the overexpression of E6 and E7 oncoproteins, which, in turn, induce p53 and pRb degradation, respectively. Additionally, it has been suggested that HR-HPV oncoproteins are involved in the regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), inducing cancer progression and metastasis. Previously, we reported that HPV16 E7 oncoprotein promotes Pirin upregulation resulting in increased epithelial-mesenchymal transition (EMT) and cell migration, with Pirin being an oxidative stress sensor and activator of NF-κB. In this study, we demonstrate the mechanism by which HPV16 E7-mediated Pirin overexpression occurs by promoting EGFR/PI3K/AKT1/NRF2 signaling, thus causing PIR/NF-κB activation in oral tumor cells. Our results demonstrate a new mechanism by which E7 contributes to oral cancer progression, proposing PIR as a potential new therapeutic target.

Protein Kinase CK2 in Cancer Energetics.

Protein kinase CK2 (formerly known as casein kinase 2) is abnormally elevated in many cancers. This may increase tumor aggressiveness through CK2-dependent phosphorylation of key proteins in several signaling pathways. In this work, we have compiled evidence from the literature to suggest that CK2 also modulates a metabolic switch characteristic of cancer cells that enhances resistance to death, due to either drugs or to a microenvironment deficient in oxygen or nutrients. Concurrently, CK2 may help to preserve mitochondrial activity in a PTEN-dependent manner. PTEN, widely recognized as a tumor suppressor, is another CK2 substrate in the PI3K/Akt signaling pathway that promotes cancer viability and aerobic glycolysis. Given that CK2 can regulate Akt as well as two of its main effectors, namely mTORC1 and ß-catenin, we comprehensively describe how CK2 may modulate cancer energetics by regulating expression of key targets and downstream processes, such as HIF-1 and autophagy, respectively. Thus, the specific inhibition of CK2 may lead to a catastrophic death of cancer cells, which could become a feasible therapeutic strategy to beat this devastating disease. In fact, ATP-competitive inhibitors, synthetic peptides and antisense oligonucleotides have been designed as CK2 inhibitors, some of them used in preclinical models of cancer, of which TBB and silmitasertib are widely known. We will finish by discussing a hypothetical scenario in which cancer cells are "addicted" to CK2; i.e., in which many proteins that regulate signaling pathways and metabolism-linked processes are highly dependent on this kinase.

Follicle-stimulating hormone promotes nerve growth factor and vascular endothelial growth factor expression in epithelial ovarian cells.

Ovarian cancer is the first cause of death for gynecological malignances in developed countries and around 80% correspond to Epithelial Ovarian Cancer (EOC). Overexpression of Nerve Growth Factor (NGF) and its high affinity receptor TRKA are involved in EOC progression, modulating several oncogenic processes such as angiogenesis by the increase of Vascular Endothelial Growth Factor (VEGF). FSH receptors (FSH-R) are present in EOC, but their changes and contribution during EOC progression are still not thoroughly known. The aims of this study were to evaluate the abundance of FSH receptors during EOC differentiation and to determine whether FSH modulates oncoproteins such as NGF and VEGF in ovarian cells. FSH-R expression in EOC tissues and cell lines (A2780, poorly differentiated EOC cells and HOSE, non-tumoral ovarian surface epithelial cells) were measured by RT-PCR and laser capture of epithelial cells from EOC samples by qPCR. FSH-R protein levels were evaluated by immunohisto/cytochemistry. Additionally, ovarian explants and ovarian cell lines were stimulated with FSH and/or FSH-R inhibitor to assess NGF and VEGF mRNA and protein levels. The results showed that FSH-R levels decreased during EOC progression, nevertheless these receptors are still present in poorly differentiated EOC. FSH increased NGF expression in ovarian cells, which was prevented using a FSH-R inhibitor. Similarly, in ovarian cancer explants, FSH increased NGF and VEGF mRNA, as well as NGF protein levels. These results suggest that FSH would display a key role not only in initial stages of EOC, but also in late stages of this disease, by modulation of NGF and VEGF levels in EOC cells.

Merkel cell polyomavirus detected in head and neck carcinomas from Chile.

BACKGROUND: The role of human polyomaviruses (HPyVs) in epithelial tumors such as head and neck carcinomas (HNSCCs) including oral and oropharyngeal carcinomas has not been established. In this study, we evaluated for the first time the presence of Merkel cell polyomavirus (MCPyV), BK human polyomavirus (BKPyV), and JC human polyomavirus (JCPyV) in HNSCCs from Chilean subjects. METHODS: One hundred and twenty HNSCCs were analyzed for the presence of MCPyV, BKPyV and JCPyV using real-time polymerase chain reaction procedures. In addition, 54 oral brushes from age- and sex-paired subjects were analyzed. RESULTS: Of the total of 120 HNSCCs, 15 were positive for MCPyV (12.5%). Only one case was positive for BKPyV (0.8%) and none for JCPyV (0%). In subjects without cancer, only one case (1.8%) resulted positive for MCPyV and none for JCPyV and BKPyV. MCPyV was associated with HNSCCs (p = 0.0239; OR = 7.571; 95% CI: 1.192-81.46). No association was found between age (p = 0.1996), gender (p = 0.7111) or differentiation status (p > 0.9999) and MCPyV presence in HNSCCs. CONCLUSIONS: MCPyVs were detected in HNSCCs from Chilean patients and were not detected in oral brushes from patients without cancer. More studies are warranted for defining an etiological role and clinical/molecular consequences of these viruses in HNSCCs.

Germline Variants in Driver Genes of Breast Cancer and Their Association with Familial and Early-Onset Breast Cancer Risk in a Chilean Population.

The genetic variations responsible for tumorigenesis are called driver mutations. In breast cancer (BC), two studies have demonstrated that germline mutations in driver genes linked to sporadic tumors may also influence BC risk. The present study evaluates the association between SNPs and SNP-SNP interaction in driver genes TTN (rs10497520), TBX3 (rs2242442), KMT2D (rs11168827), and MAP3K1 (rs702688 and rs702689) with BC risk in BRCA1/2-negative Chilean families. The SNPs were genotyped in 489 BC cases and 1078 controls by TaqMan Assay. Our data do not support an association between rs702688: A>G or rs702689: G>A and BC risk. The rs10497520-T allele was associated with a decreased risk in patients with family history of BC or early-onset BC (OR = 0.6, p < 0.0001 and OR = 0.7, p = 0.05, respectively). rs2242442-G was associated with a protective effect and rs11168827-C was associated with increased BC risk in families with a strong history of BC (OR = 0.6, p = 0.02 and OR = 1.4, p = 0.05, respectively). As rs10497520-T and rs2242442-G seemed to protect against BC risk, we then evaluated their combined effect. Familial BC risk decreased in a dose-dependent manner with the protective allele count, reflecting an additive effect (p-trend < 10-4). To our knowledge, this is the first association study of BC driver gene germline variations in a Chilean population.

Endothelin-converting enzyme-1c promotes stem cell traits and aggressiveness in colorectal cancer cells.

Endothelin-1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin-1 rely on its activation by endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N-terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R ) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self-renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2-dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC-like traits. Therefore, phospho-ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.

Curcumin decreases epithelial­mesenchymal transition by a Pirin­dependent mechanism in cervical cancer cells.

Curcumin is a natural antioxidant polyphenol, which decreases epithelial­mesenchymal transition (EMT) and cell migration in cervical cancer cells. However, the mechanism by which such a decrease occurs is unclear. It is well established that cervical cancer can be caused by high­risk human papillomavirus (HPV), which overexpresses E6 and E7 oncoproteins. Recent findings have suggested that viral oncoproteins regulate the expression of Pirin, which is an oxidative stress sensor involved in EMT and cell migration. Molecular markers associated with EMT, pirin and HPV were evaluated using reverse transcription­reverse quantitative PCR and western blotting. In addition, the migratory ability of cells was evaluated using a Transwell assay. In order to evaluate the role of Pirin in curcumin­mediated inhibition of EMT, SiHa cervical carcinoma cells, which contain two integrated copies of HPV16, were exposed to curcumin. Cell migration, and the expression levels of EMT biomarkers and the pirin protein, which is a product of the PIR gene, were subsequently evaluated. The results demonstrated a significant decrease in EMT following exposure to 20 µM curcumin for 72 h. This finding was supported by a decrease in the protein expression levels of N­cadherin, Vimentin and Slug. Furthermore, it was observed that PIR expression and Pirin protein levels were significantly decreased when SiHa cells were exposed to curcumin. Subsequently, to analyze the effects of Pirin on EMT, SiHa cells were transfected with a small interfering RNA (siRNA) to knockdown PIR. A significant increase in E­cadherin mRNA expression and a decrease in N­cadherin protein expression were observed. In addition, a similar decrease was observed when SiHa cells were exposed to both PIR siRNA and curcumin. Finally, a significant decrease in SiHa cell migration was observed in the presence of 20 µM curcumin compared with in the control group. These findings suggested that curcumin may decrease EMT, at least in part by a Pirin­dependent mechanism. Therefore, Pirin protein may be an important pharmacological target for cervical cancer treatment.

Endothelin-converting enzyme-1 in cancer aggressiveness.

The endothelin-1 (ET-1) axis contributes to the pathophysiology of several cancers by promoting tumor development and progression. This peptide is activated from its precursor, big ET-1, by endothelin-converting enzyme-1 (ECE-1). Active ET-1 binds to its cognate G-coupled receptor, ETAR, which transduces the signal to the inside of the cell. ET-1 has a short half-life of about 90 s, so its biological effects are completely dependent on its enzymatic activation by ECE-1. Expression of ECE-1 is elevated in several tumors and cancer cell lines. There are four ECE-1 isoforms -ECE-1a, -1b, -1c, and -1d- which vary in terms of their subcellular localization and, in some cases, their effects on cancer-related properties such as proliferation and invasiveness. In this article, we review findings on the role of ECE-1, particularly isoform ECE-1c, in oncogenesis and malignant progression. We also review evidence regarding ECE-1 expression in several types of tumors and cancer cell lines. Recent findings from our laboratory and others allow us to speculate on the mechanism by which ECE-1c promotes cancer aggressiveness. Finally, we evaluate potential post-translational modifications of ECE-1c, highlighting phosphorylation by several kinases, as well as evidence pointing to a putative, non-canonical, ET-1-independent mechanism for promoting invasiveness. Taken together, current evidence suggests that ECE-1c contributes to cancer aggressiveness and plays a putative role as a key regulator of cancer progression. Therefore, we propose that this protein is a promising target for prognostic and therapeutic purposes.