Identification of non-small cell lung cancer biomarkers using mass spectrometry

Comunicaciones a congreso

Synergistic effect of antimetabolic and chemotherapy drugs in triple-negative breast cancer

The triple-negative breast cancer (TNBC) subtype comprises approximately 15% of all breast cancers and is associated with poor long-term outcomes. Classical chemotherapy remains the standard of treatment, with toxicity and resistance being major limitations. TNBC is a high metabolic group, and antimetabolic drugs are effective in inhibiting TNBC cell growth. We analyzed the combined effect of chemotherapy and antimetabolic drug combinations in MDA-MB-231, MDA-MB-468 and HCC1143 human TNBC cell lines. Cells were treated with each drug or with drug combinations at a range of concentrations to establish the half-maximal inhibitory concentrations (IC50). The dose-effects of each drug or drug combination were calculated, and the synergistic or antagonistic effects of drug combinations were defined. Chemotherapy and antimetabolic drugs exhibited growth inhibitory effects on TNBC cell lines. Antimetabolic drugs targeting the glycolysis pathway had a synergistic effect with chemotherapy drugs, and antiglycolysis drug combinations also had a synergistic effect. The use of these drug combinations could lead to new therapeutic strategies that reduce chemotherapy drug doses, decreasing their toxic effect, or that maintain the doses but enhance their efficacy by their synergistic effect with other drugsMaría I. Lumbreras-Herrera and Andrea Zapater-Moros are supported by Consejería de Educación e Investigación de la Comunidad de Madrid (IND2018/BMD-9262). Elena López-Camacho is supported by the Spanish Economy and Competitiveness Ministry (PTQ2018–009760). This work is supported by an unrestricted grant from Roch

A novel molecular analysis approach in colorectal cancer suggests new treatment opportunities

Colorectal cancer (CRC) is a molecular and clinically heterogeneous disease. In 2015, the Colorectal Cancer Subtyping Consortium classified CRC into four consensus molecular subtypes (CMS), but these CMS have had little impact on clinical practice. The purpose of this study is to deepen the molecular characterization of CRC. A novel approach, based on probabilistic graphical models (PGM) and sparse k-means–consensus cluster layer analyses, was applied in order to functionally characterize CRC tumors. First, PGM was used to functionally characterize CRC, and then sparse k-means–consensus cluster was used to explore layers of biological information and establish classifications. To this aim, gene expression and clinical data of 805 CRC samples from three databases were analyzed. Three different layers based on biological features were identified: adhesion, immune, and molecular. The adhesion layer divided patients into high and low adhesion groups, with prognostic value. The immune layer divided patients into immune-high and immunelow groups, according to the expression of immune-related genes. The molecular layer established four molecular groups related to stem cells, metabolism, the Wnt signaling pathway, and extracellular functions. Immune-high patients, with higher expression of immune-related genes and genes involved in the viral mimicry response, may benefit from immunotherapy and viral mimicry-related therapies. Additionally, several possible therapeutic targets have been identified in each molecular group. Therefore, this improved CRC classification could be useful in searching for new therapeutic targets and specific therapeutic strategies in CRC diseas

An 8-gene qRT-PCR-based gene expression score that has prognostic value in early breast cancer

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling may improve prognostic accuracy in patients with early breast cancer. Our objective was to demonstrate that it is possible to develop a simple molecular signature to predict distant relapse.</p> <p>Methods</p> <p>We included 153 patients with stage I-II hormonal receptor-positive breast cancer. RNA was isolated from formalin-fixed paraffin-embedded samples and qRT-PCR amplification of 83 genes was performed with gene expression assays. The genes we analyzed were those included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Index. The association among gene expression, clinical variables and distant metastasis-free survival was analyzed using Cox regression models.</p> <p>Results</p> <p>An 8-gene prognostic score was defined. Distant metastasis-free survival at 5 years was 97% for patients defined as low-risk by the prognostic score versus 60% for patients defined as high-risk. The 8-gene score remained a significant factor in multivariate analysis and its performance was similar to that of two validated gene profiles: the 70-Gene Signature and the Recurrence Score. The validity of the signature was verified in independent cohorts obtained from the GEO database.</p> <p>Conclusions</p> <p>This study identifies a simple gene expression score that complements histopathological prognostic factors in breast cancer, and can be determined in paraffin-embedded samples.</p

Utility of CYP2D6 copy number variants as prognostic biomarker in localized anal squamous cell carcinoma

Background: Anal squamous cell carcinoma (ASCC) is an infrequent tumor whose treatment has not changed since the 1970s. The aim of this study is the identification of biomarkers allowing personalized treatments and improvement of therapeutic outcomes. Methods: Forty-six paraffin tumor samples from ASCC patients were analyzed by whole-exome sequencing. Copy number variants (CNVs) were identified and their relation to disease-free survival (DFS) was studied and validated in an independent retrospective cohort of 101 ASCC patients from the Multidisciplinary Spanish Digestive Cancer Group (GEMCAD). GEMCAD cohort proteomics allowed assessing the biological features of these tumors. Results: On the discovery cohort, the median age was 61 years old, 50% were males, stages I/II/III: 3 (7%)/16 (35%)/27 (58%), respectively, median DFS was 33 months, and overall survival was 45 months. Twenty-nine genes whose duplication was related to DFS were identified. The most representative was duplications of the CYP2D locus, including CYP2D6, CYP2D7P, and CYP2D8P genes. Patients with CYP2D6 CNV had worse DFS at 5 years than those with two CYP2D6 copies (21% vs. 84%; p <.0002, hazard ratio [HR], 5.8; 95% confidence interval [CI], 2.7–24.9). In the GEMCAD validation cohort, patients with CYP2D6 CNV also had worse DFS at 5 years (56% vs. 87%; p =.02, HR = 3.6; 95% CI, 1.1–5.7). Mitochondria and mitochondrial cell-cycle proteins were overexpressed in patients with CYP2D6 CNV. Conclusions: Tumor CYP2D6 CNV identified patients with a significantly worse DFS at 5 years among localized ASCC patients treated with 5-fluorouracil, mitomycin C, and radiotherapy. Proteomics pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets for these high-risk patients. Plain Language Summary: Anal squamous cell carcinoma is an infrequent tumor whose treatment has not been changed since the 1970s. However, disease-free survival in late staged tumors is between 40% and 70%. The presence of an alteration in the number of copies of CYP2D6 gene is a biomarker of worse disease-free survival. The analysis of the proteins in these high-risk patients pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets. Therefore, the determination of the number of copies of CYP2D6 allows the identification of anal squamous carcinoma patients with a high-risk of relapse that could be redirected to a clinical trial. Additionally, this study may be useful to suggest new treatment strategies to increase current therapy efficacyIdiPAZ, Grant/Award Number: Jesús Antolín Garciarena Fellowship; European Proteomics Infrastructure Consortium, Grant/Award Number: 823839, Horizon 2020 Programm

GRP78 Knockdown Enhances Apoptosis via the Down-Regulation of Oxidative Stress and Akt Pathway after Epirubicin Treatment in Colon Cancer DLD-1 Cells

INTRODUCTION: The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway. CONCLUSIONS: Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells

CCR9-CCL25 interactions promote cisplatin resistance in breast cancer cell through Akt activation in a PI3K-dependent and FAK-independent fashion

<p>Abstract</p> <p>Background</p> <p>Chemotherapy heavily relies on apoptosis to kill breast cancer (BrCa) cells. Many breast tumors respond to chemotherapy, but cells that survive this initial response gain resistance to subsequent treatments. This leads to aggressive cell variants with an enhanced ability to migrate, invade and survive at secondary sites. Metastasis and chemoresistance are responsible for most cancer-related deaths; hence, therapies designed to minimize both are greatly needed. We have recently shown that CCR9-CCL25 interactions promote BrCa cell migration and invasion, while others have shown that this axis play important role in T cell survival. In this study we have shown potential role of CCR9-CCL25 axis in breast cancer cell survival and therapeutic efficacy of cisplatin.</p> <p>Methods</p> <p>Bromodeoxyuridine (BrdU) incorporation, Vybrant apoptosis and TUNEL assays were performed to ascertain the role of CCR9-CCL25 axis in cisplatin-induced apoptosis of BrCa cells. Fast Activated Cell-based ELISA (FACE) assay was used to quantify <it>In situ </it>activation of PI3K^p85, Akt^Ser473, GSK-3β^Ser9and FKHR^Thr24in breast cancer cells with or without cisplatin treatment in presence or absence of CCL25.</p> <p>Results</p> <p>CCR9-CCL25 axis provides survival advantage to BrCa cells and inhibits cisplatin-induced apoptosis in a PI3K-dependent and focal adhesion kinase (FAK)-independent fashion. Furthermore, CCR9-CCL25 axis activates cell-survival signals through Akt and subsequent glycogen synthase kinase-3 beta (GSK-3β) and forkhead in human rhabdomyosarcoma (FKHR) inactivation. These results show that CCR9-CCL25 axis play important role in BrCa cell survival and low chemotherapeutic efficacy of cisplatin primarily through PI3K/Akt dependent fashion.</p

Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Despite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, <it>TSC1 </it>and <it>TSC2</it>, and other members of this pathway in tumorigenesis of OSCC.</p> <p>Methods</p> <p>Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the <it>TSC1</it>, <it>TSC2 </it>and <it>PTEN </it>candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the <it>TSC2 </it>promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).</p> <p>Results</p> <p>The semi-quantitative RT-PCR analysis showed downregulation of <it>TSC1</it>, <it>TSC2</it>, <it>EIF4EBP1 </it>and <it>PTEN</it>, and upregulation of <it>PIK3C2A</it>, <it>AKT1</it>, <it>PDPK1</it>, <it>RHEB</it>, <it>FRAP1</it>, <it>RPS6KB1</it>, <it>EIF4E </it>and <it>RPS6 </it>in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the <it>TSC2 </it>gene only. In order to confirm the results in patient tumor samples, the methylation status of the <it>TSC2 </it>gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.</p> <p>Conclusion</p> <p>Our study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.</p

Association of prolactin receptor (PRLR) variants with prolactinomas

Prolactinomas are the most frequent type of pituitary tumors, which represent 10–20% of all intracranial neoplasms in humans. Prolactinomas develop in mice lacking the prolactin receptor (PRLR), which is a member of the cytokine receptor superfamily that signals via Janus kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) or phosphoinositide 3-kinase-Akt (PI3K-Akt) pathways to mediate changes in transcription, differentiation and proliferation. To elucidate the role of the PRLR gene in human prolactinomas, we determined the PRLR sequence in 50 DNA samples (35 leucocytes, 15 tumors) from 46 prolactinoma patients (59% males, 41% females). This identified six germline PRLR variants, which comprised four rare variants (Gly57Ser, Glu376Gln, Arg453Trp and Asn492Ile) and two low-frequency variants (Ile76Val, Ile146Leu), but no somatic variants. The rare variants, Glu376Gln and Asn492Ile, which were in complete linkage disequilibrium, and are located in the PRLR intracellular domain, occurred with significantly higher frequencies (P 1.3-fold, P < 0.02) and proliferation (1.4-fold, P < 0.02), but did not affect pSTAT5 signaling. Treatment of cells with an Akt1/2 inhibitor or everolimus, which acts on the Akt pathway, reduced Asn492Ile signaling and proliferation to WT levels. Thus, our results identify an association between a gain-of-function PRLR variant and prolactinomas and reveal a new etiology and potential therapeutic approach for these neoplasms