Jaime A. Espinoza

Molecular classification of gastric cancer: Towards a pathway-driven targeted therapy

Gastric cancer (GC) is the third leading cause of cancer mortality worldwide. Although surgical resection is a potentially curative approach for localized cases of GC, most cases of GC are diagnosed in an advanced, non-curable stage and the response to traditional chemotherapy is limited. Fortunately, recent advances in our understanding of the molecular mechanisms that mediate GC hold great promise for the development of more effective treatment strategies. In this review, an overview of the morphological classification, current treatment approaches, and molecular alterations that have been characterized for GC are provided. In particular, the most recent molecular classification of GC and alterations identified in relevant signaling pathways, including ErbB, VEGF, PI3K/AKT/mTOR, and HGF/MET signaling pathways, are described, as well as inhibitors of these pathways. An overview of the completed and active clinical trials related to these signaling pathways are also summarized. Finally, insights regarding emerging stem cell pathways are described, and may provide additional novel markers for the development of therapeutic agents against GC. The development of more effective agents and the identification of biomarkers that can be used for the diagnosis, prognosis, and individualized therapy for GC patients, have the potential to improve the efficacy, safety, and cost-effectiveness for GC treatments.

Mitochondrial membrane potential disruption pattern in human sperm

BACKGROUND Loss of mitochondrial membrane potential (DeltaPsi(m)) in spermatozoa is correlated with high levels of reactive oxygen species in semen, abnormal spermiogram parameters, and low success rates of IVF. In somatic cells, the loss of DeltaPsi(m) is primarily associated with several mechanisms of cell death, mainly the activation of caspases. The impact of mitochondrial dysfunction on sperm function is still not fully elucidated, although disruption of DeltaPsi(m) and activation of caspases are processes thoroughly studied in human ejaculates. Disruption of DeltaPsi(m) in sperm can be externally triggered by the antineoplastic agent betulinic acid (BA). In this study, we determined whether caspase activation is necessary for the BA-induced disruption of DeltaPsi(m) in human sperm. METHODS Viable and highly motile sperm cells were selected through a swim-up process and incubated with 90 microg/ml BA. To elucidate the caspase dependency of BA-triggered disruption of DeltaPsi(m), we used the pan-caspase inhibitor zVAD-fmk and the caspase-3/7 inhibitor DEVD-cho. RESULTS Exposing highly motile sperm to BA caused a specific disruption of DeltaPsi(m) (P < 0.001 versus control) and a corresponding increase in caspase-3/7 activity (P < 0.001 versus control). Pre-incubation of the sperm with zVAD-fmk or DEVD-cho only partially inhibited BA-induced loss of DeltaPsi(m) (P < 0.05 versus control). CONCLUSION We found that caspases directly participate in the loss of DeltaPsi(m) caused by BA in human sperm cells. However, caspase-independent pathways may also be present.

miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in gastric cancer through regulation of the PI3K/AKT/mTOR pathway

BackgroundGastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs.MethodsTwenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells.ResultsmiR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively.ConclusionsOur expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.

Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclophosphamide and gene transfer of IL-12

Immunotherapy‐based strategies for gastrointestinal carcinomas (GIC) have been exploited so far, but these approaches have to face strong mechanisms of immune escape induced by tumours. We previously demonstrated that sub‐therapeutic doses of an adenovirus expressing IL‐12 genes (AdIL‐12) mediated a potent antitumour effect against subcutaneous (s.c.) colorectal carcinomas (CRC) in mice pre‐treated with low doses of cyclophosphamide (Cy). In our study we used this combination to assess its impact on the immunosuppressive microenvironment. In s.c. CRC model we demonstrated that non‐responder mice failed to decrease Tregs in tumour, spleen and peripheral blood. Reconstitution of Tregs into tumour‐bearing mice treated with combined therapy abolished the antitumoural effect. In addition, Cy + AdIL‐12 modified Tregs functionality by inhibiting the in vitro secretion of IL‐10 and TGF‐β and their ability to inhibit dendritic cells activation. Combined treatment decreased the number of myeloid‐derived suppressor cells (MDSCs) in comparison to non‐treated mice and, interestingly, administration of Tregs restored splenic MDSCs population. Furthermore, combined therapy potently generated specific cytotoxic IFN‐γ‐secreting CD4+ T cells able to eradicate established CRC tumours after adoptive transfer. Finally, we evaluated the combination on disseminated CRC and pancreatic carcinoma (PC). Cy + AdIL‐12 were able to eradicate liver metastatic CRC (47%) and PC tumour nodules (40%) and to prolong animal survival. The results of this study support the hypothesis that Cy + AdIL‐12 might be a valid immunotherapeutic strategy for advanced GIC.

Targeting specific molecular pathways holds promise for advanced gallbladder cancer therapy

Gallbladder cancer is the most common and aggressive malignancy of the biliary tract. The complete surgical resection is the only potentially curative approach in early stage; however, most cases are diagnosed in advanced stages and the response to traditional chemotherapy and radiotherapy is extremely limited, with modest impact in overall survival. The recent progress in understanding the molecular alterations of gallbladder cancer has shown great promise for the development of more effective treatment strategies. This has mainly resulted from the identification of molecular alterations in relevant intracellular signaling pathways-Hedgehog, PI3K/AKT/mTOR, Notch, ErbB, MAPK and angiogenesis-which are potential tailored targets for gallbladder cancer patients. This review discusses the recent remarkable progress in understanding the molecular alterations that represent novel prognosis molecular markers and therapeutic targets for gallbladder cancer, which will provide opportunities for research and for developing innovative strategies that may enhance the benefit of conventional chemotherapy, or eventually modify the fatal natural history of this orphan disease.

The low‐abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer

Studies on the low‐abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low‐abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low‐abundance transcriptome and the whole transcriptome. Analysis of the low‐abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type‐specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF‐C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low‐abundance transcriptome provides useful insights into the molecular basis and treatment of cancer.

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines.

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.

FABP7 and HMGCS2 are novel protein markers for apocrine differentiation categorizing apocrine carcinoma of the breast.

Apocrine carcinoma of the breast is a distinctive malignancy with unique morphological and molecular features, generally characterized by being negative for estrogen and progesterone receptors, and thus not electable for endocrine therapy. Despite the fact that they are morphologically distinct from other breast lesions, no standard molecular criteria are currently available for their diagnosis. Using gel-based proteomics in combination with mass spectrometry and immunohistochemistry we have identified two novel markers, HMGCS2 and FABP7 that categorize the entire breast apocrine differentiation spectrum from benign metaplasia and cysts to invasive stages. Expression of HMGCS2 and FABP7 is strongly associated with apocrine differentiation; their expression is retained by most invasive apocrine carcinomas (IAC) showing positive immunoreactivity in 100% and 78% of apocrine carcinomas, respectively, as compared to non-apocrine tumors (16.7% and 6.8%). The nuclear localization of FABP7 in tumor cells was shown to be associated with more aggressive stages of apocrine carcinomas. In addition, when added to the panel of apocrine biomarkers previously reported by our group: 15-PGDH, HMGCR and ACSM1, together they provide a signature that may represent a golden molecular standard for defining the apocrine phenotype in the breast. Moreover, we show that combining HMGCS2 to the steroidal profile (HMGCS2+/Androgen Receptor (AR)+/Estrogen Receptor(ER)-/Progesteron Receptor (PR)- identifies IACs with a greater sensitivity (79%) as compared with the steroidal profile (AR+/ER-/PR-) alone (54%). We have also presented a detailed immunohistochemical analysis of breast apocrine lesions with a panel of antibodies against proteins which correspond to 10 genes selected from published transcriptomic signatures that currently characterize molecular apocrine subtype and shown that except for melanophilin that is overexpressed in benign apocrine lesions, these proteins were not specific for morphological apocrine differentiation in breast.

Low expression of equilibrative nucleoside transporter 1 is associated with poor prognosis in chemotherapy‐naïve pT2 gallbladder adenocarcinoma patients

Equilibrative nucleoside transporter 1 (ENT1) is the major transporter of the chemotherapeutic drug gemcitabine, the current therapy for advanced gallbladder cancer (GBC). ENT1 expression has been proposed as a predictive marker for gemcitabine‐treated pancreatic cancer patients. The aim of study was to explore the value of ENT1 measurement in chemotherapy‐naïve patients with advanced GBC.

Rapamycin and WYE-354 suppress human gallbladder cancer xenografts in mice

Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.