Helga Weber

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.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

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.

AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines

Background Gallbladder carcinoma is a highly malignant tumor and a public health problem in some parts of the world. It is characterized by a poor prognosis and its resistance to radio and chemotherapy. There is an urgent need to develop novel therapeutic alternatives for the treatment of gallbladder carcinoma. The mammalian target of the rapamycin (mTOR) signaling pathway is activated in about 50% of human malignancies, and its role in gallbladder carcinoma has previously been suggested. In the present study, we investigated the phosphorylation status of the mTOR substrate p70S6K in preneoplastic and neoplastic gallbladder tissues and evaluated the effect of three mTOR inhibitors on cell growth and migration in gallbladder carcinoma cell lines. Methods Immunohistochemical staining of phospho-p70S6K was analyzed in 181 gallbladder carcinoma cases, classified according to lesion type as dysplasia, early carcinoma, or advanced carcinoma. Protein expression of AKT/mTOR members was also evaluated in eight gallbladder carcinoma cell lines by Western blot analysis. We selected two gallbladder carcinoma cell lines (G415 and TGBC-2TKB) to evaluate the effect of rapamycin, RAD001, and AZD8055 on cell viability, cell migration, and protein expression. Results Our results showed that phospho-p70S6K is highly expressed in dysplasia (66.7%, 12/18), early cancer (84.6%, 22/26), and advanced cancer (88.3%, 121/137). No statistical correlation was observed between phospho-p70S6K status and any clinical or pathological features, including age, gender, ethnicity, wall infiltration level, or histological differentiation (P < 0.05). In vitro treatment with rapamycin, RAD001, and AZD8055 reduced cell growth, cell migration, and phospho-p70S6K expression significantly in G-415 and TGBC-2TKB cancer cells (P < 0.001). Conclusion Our findings confirm the upregulation of this signaling pathway in gallbladder carcinoma and provide a rationale for the potential use of mTOR inhibitors as a therapeutic strategy for human gallbladder carcinoma.

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.

A Novel CDC25B Promoter–Based Oncolytic Adenovirus Inhibited Growth of Orthotopic Human Pancreatic Tumors in Different Preclinical Models

Purpose: We decided to construct a novel oncolytic adenovirus whose replication was driven by the CDC25B promoter for its use in preclinical models of pancreatic cancer. Experimental Design: We placed the essential E1A gene under control of the CDC25B promoter. Based on preliminary data, we pseudotyped the adenovirus with a chimeric fiber of serotypes 5/3. We investigated the in vitro lytic effect and the in vivo therapeutic efficacy in combination with gemcitabine on human pancreatic tumor xenografts orthotopically growing in nude mice and in tumors growing in Syrian hamsters. We also assessed biochemical markers of hepatic toxicity and CA19.9 levels. Results: AV25CDC exhibited a strong in vitro lytic effect on pancreatic cancer cells. In vivo administration of AV25CDC combined with gemcitabine in mice harboring subcutaneously growing SW1990 pancreatic tumors almost abrogated tumor growth. Nude mice harboring 15-day-old orthotopic tumors, treated intratumorally or systemically with AV25CDC combined with gemcitabine, exhibited 70% to 80% reduction in tumor size compared with control mice that lasted for at least 60 days. Chemovirotherapy treatment induced a return to normal levels of biochemical parameters of hepatic toxicity; these mice exhibited more than 90% reduction in CA19.9 serum levels compared with control. Chemovirotherapy efficacy was confirmed in mice harboring Mia PaCa-2 tumors and in Syrian hamster harboring HaP-T1 tumors. We observed that viral treatment disrupted tumor architecture and induced an increase in MMP-9 activity that might facilitate gemcitabine penetrability. Conclusion: These data demonstrate that AV25CDC is an effective oncolytic agent candidate for pancreatic cancer chemovirotherapy combination. Clin Cancer Res; 21(7); 1665–74. ©2015 AACR.

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.

Efectos de la Inhibición de C-Flipl en Líneas Celulares de Cáncer de Cuello Uterino

Cuando las variantes Short y Raji de la proteina Cellular FLICE-like inhibitory protein (c-FLIP) se encuentran sobrexpresadas son capaces de inhibir la apoptosis, mientras la funcion de la isoforma Long (c-FLIPL), depende de la concentracion de esta molecula en las celulas. El objetivo de este estudio fue determinar los efectos de la inhibicion de c-FLIPL en lineas celulares de cancer de cuello uterino. Para realizar el estudio fueron utilizadas SiHa, C-4I y C-33A, lineas celulares de cancer cervical. La expresion de c-FLIPL en estas lineas fue establecida mediante PCR en tiempo real y western blot. Posteriormente la expresion de c-FLIPL fue inhibida, mediante transfecion transiente con siRNA complementario al mRNA mensajero de c.-FLIPL. Los efectos de esta inhibicion en la viabilidad celular, proliferacion y apoptosis fue comparada con celulas transfectadas con un siRNA control (scrambled). Una vez reprimido c-FLIPL, las lineas celulares SiHa y C-4I presentaron un aumento de la viabilidad celular (P<0,05). Para evaluar la proliferacion celular se utilizo inmunocitoquimica de los marcadores Ki-67 y PCNA. Las celulas SiHa transfectadas con siRNA c-FLIPL, mostraron una elevada expresion de Ki-67 (P<0,0001), mientras que las celulas C-33A con c-FLIPL inhibido mostraron una menor expresion de PCNA (P<0,01). Las tres lineas celulares con c-FLIPL reprimido mostraron un mayor nivel de apoptosis que las celulas control. Estos resultados sugieren que c-FLIPL puede tener efectos en la proliferacion y apoptosis de lineas celulares de cancer de cuello uterino.

Abstract 647: Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC New biomarkers are needed to improve cervical cancer screening technologies, which are mostly based on cytological examination and HPV detection. However, PAP-smear has a low-sensitivity to detect low grade squamous intraepithelial lesions and not all HPV infected women will develop preneoplastic or neoplastic lesions. Previous results of our group showed that genes Gen Z (patent pending), CDH1 and MEGF9 could be hypermethylated in cervical cancer and not in normal epithelia. The aim of this study was to determinate if promoter methylation status of genes (Gen Z, CDH1 and MEGF9) are related with progression or diagnosis of cervical carcinogenesis. For this study, 107 citobrush, urine and blood samples were collected from women who attended to gynecological care in a public health center in Temuco, Chile. Citobrush DNA from 50 normal, 40 low grade squamous intraepithelial lesion (LSIL), 40 high grade squamous intraepithelial lesion (HSIL) and 17 squamous cervical cancer were bisulfite converted for methylation specific PCR (MSP). Bisulfite conversion was confirmed by amplification of a 133-bp fragment of the β-actin. MSP primers were specifically design for CpG island of promotor region of each gene. Gen Z was found 100% methylated in SCC samples, 65% in HSIL, 43% in LSIL and in normal samples only a 26%. MEGF9 and CDH1 genes were found methylated in 36% and 48% of normal samples, 45% and 55% of LSIL, 70% and 77% of HSIL and 47% and 71% of SCC, respectively. All promoter regions studied showed a higher methylation frequency in LSIL, HSIL and SCC than normal samples. Significant statistical differences in Gen Z and CDH1 Methylation frequencies between normal and SCC samples were found (P<0.05). Methylation of Gen Z increased in a sequential and cumulative way as the lesion progress. Our results suggest that the Gen Z could be useful tool for identifying women with a higher risk of progression to cervical cancer. Examination of these biomarkers in a larger, independent cohort is warranted. Grant Support: This investigation was financed by Proyect CORFO-INNOVA N°07CN13PBT-222 and Proyect CORFO N° 09CN14-5960 (CEGIN). CI is recipient of grants from FONDECYT Postdoctoral Proyect N° 3130630. PB is recipient of grants from FONDECYT Postdoctoral Proyect N° 3120141 Citation Format: Priscilla Brebi, Alejandra Andana, Rene Hoffstetter, Carmen Ili, Tamara Viscarra, Ramon Silva, Patricia Garcia, Pamela Leal, Helga Weber, Juan C. Roa. Novel promoter hypermethylation marker for prognostic in cervicouterine cancinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 647. doi:10.1158/1538-7445.AM2013-647