Carolina Bizama

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.

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.

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 inflammatory inception of gallbladder cancer.

Gallbladder cancer is a lethal disease with notable geographical variations worldwide and a predilection towards women. Its main risk factor is prolonged exposure to gallstones, although bacterial infections and other inflammatory conditions are also associated. The recurrent cycles of gallbladder epithelium damage and repair enable a chronic inflammatory environment that promotes progressive morphological impairment through a metaplasia-dysplasia-carcinoma, along with cumulative genome instability. Inactivation of TP53, which is mutated in over 50% of GBC cases, seems to be the earliest and one of the most important carcinogenic pathways involved. Increased cell turnover and oxidative stress promote early alteration of TP53, cell cycle deregulation, apoptosis and replicative senescence. In this review, we will discuss evidence for the role of inflammation in gallbladder carcinogenesis obtained through epidemiological studies, genome-wide association studies, experimental carcinogenesis, morphogenetic studies and comparative studies with other inflammation-driven malignancies. The evidence strongly supports chronic, unresolved inflammation as the main carcinogenic mechanism of gallbladder cancer, regardless of the initial etiologic trigger. Given this central role of inflammation, evaluation of the potential for GBC prevention removing causes of inflammation or using anti-inflammatory drugs in high-risk populations may be warranted.

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.

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

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.

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors. In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies. In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05). The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%). Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

Abstract 4267: Establishment and biological characterization of a Chilean ascites-derived gallbladder cancer cell line

Gallbladder cancer is the most common biliary tract cancer. Globally, it is considered as a rare malignancy but shows a high incidence in certain geographic areas, such as Eastern and South Asia, Eastern Europe and Latin America. In Chile, this aggressive neoplasia is the second leading cause of cancer death among women, with a mortality rate of 15 deaths per 100.000 women, only slightly lower than breast cancer. Most patients with gallbladder cancer are diagnosed at advanced stages and the prognosis still remains low, even using the most current diagnostic techniques. Tumor biology of gallbladder cancer is still poorly understood and there are no therapeutic options to improve the prognosis of patients with advanced gallbladder cancer. The establishment of cell lines for their use as in vitro models is essential for the study of tumor biology and drug susceptibility. The aim of this study was to characterize and compare the malignant properties of three gallbladder cancer clones isolated from ascites-derived primary culture cells. Tumor cells were isolated from the ascites of an advanced gallbladder cancer patient using a previously established protocol. The primary culture cells were characterized to determine their epithelial origin by using immunohistochemical markers. Due to the heterogeneous nature of these cells, individual clones were isolated from them and maintained in culture until their establishment as immortal cell lines (less than 20 passages). Finally, three clones were obtained and evaluated in order to characterize and compare their malignant properties, determining their growth rate, chemosensitivity to gemcitabine, cisplatin and 5-fluoracil, migration capability and the in vivo tumorigenesis induction. The ascites-derived primary culture cells showed high expression of epithelial and tumor markers (Cytokeratin 7 and 19, CA 19-9, CA 125, CA 15-3) and negative expression of mesothelial markers (calretinin and mesothelin). Individual clones (clone 1, 2 and 3) derived from the primary culture showed differences from each other. The calculated doubling time was 60h for Clone 1, 35h for Clone 2, and 28h for Clone 3. All three clones were equally sensible to gemcitabine, cisplatin and 5-Fluoracil, compared to other established gallbladder cancer cell lines. Clone 5 exhibited the greater migration potential and Clone 6 resulted to have the most tumorigenic capability, although all were able to form xenograft tumors before 2 weeks. Conclusions: To our knowledge, these are the first gallbladder cancer cell lines established from a Chilean patient and they may provide a useful tool for the study of gallbladder cancer biology and for in vitro and in vivo analysis aimed at identification of new potential therapeutic targets. Research supported by FONDECYT 11130515, 1151008, 1130204, 3140426 and 3140308. Citation Format: Javier Retamal, Carolina Bizama, Jaime Espinoza, Lorena Rosa, Francisca Alfaro, Diego Romero, Maria Jose Apud, Bruno Nervi, Pamela Leal, Helga Weber, Juan Carlos Roa, Patricia Garcia. Establishment and biological characterization of a Chilean ascites-derived gallbladder cancer cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4267.