Andrey Frolov

ErbB3 expression and dimerization with EGFR influence pancreatic cancer cell sensitivity to erlotinib.

Abnormal expression and signaling of ErbB receptors has been implicated in multiple epithelial malignancies, including pancreatic cancer. Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has been recently approved for pancreatic cancer treatment, but there are no reliable predictors of patient response. Expression of additional ErbB receptors seems to influence tumor response to EGFR-targeted therapy. We analyzed the influence of ErbB3 expression on pancreatic cancer cell response to erlotinib treatment. Proliferation assays of five human pancreatic cancer cell lines were performed following treatment with erlotinib. Expression and phosphorylation profiles of ErbB receptors and downstream adaptor protein (Akt, ERK1/2, STAT3, mTOR) were evaluated following stimulation with EGF or neuregulin-β. The formation of EGFR homodimers and EGFR-ErbB3 heterodimers, necessary to enable ErbB3 downstream signaling, was demonstrated by chemical cross-linking assays. The effects of RNA inhibition of ErbB3 on sensitivity to erlotinib treatment were evaluated in AsPC-1 pancreatic cancer cells. Erlotinib inhibited Akt phosphorylation and proliferation of all the ErbB3-expressing cell lines but did not affect mTOR activation. Cross-linking studies confirmed the presence of EGFR-ErbB3 heterodimers in pancreatic cancer cells. Only the ErbB3-deficient MIA PaCa-2 cells displayed persistent Akt activation and ongoing proliferation in spite of erlotinib treatment. siRNA–mediated inhibition of ErbB3 expression in AsPC-1 cells resulted in acquired resistance to erlotinib treatment. Pancreatic cancer cells which lack ErbB3 do not display activation of the ErbB3-PI3K-Akt cascade induced by EGFR/ErbB3 heterodimers and become less critically dependent on EGFR signaling and therefore resistant to erlotinib. Pancreatic cancer expression of ErbB3 may be useful for EGFR-targeted therapy patient selection.

Tumor-associated down-regulation of 15-lipoxygenase-1 is reversed by celecoxib in colorectal cancer.

Objective:To evaluate the role of celecoxib on 15-lipoxygenase-1 (15-LOX-1) expression, protein levels, and rates of apoptosis in colorectal cancer cell lines. Also, to evaluate the expression of 15-LOX-1 in human normal mucosa, adenoma, and carcinoma with correlation to overall survival. Summary Background Data:The function of 15-LOX-1 is to maintain normal rates of apoptosis (programmed cell death). Decreased apoptosis is one mechanism of cancer growth and dissemination. It is our hypothesis that expression of 15-LOX-1 is reduced in human colorectal cancer (CRC) and the administration of celecoxib can reverse this process and induce apoptosis. Methods:Effect of celecoxib in cell culture: The effect of 40 μmol/L celecoxib was compared with untreated controls in tissue culture utilizing HT-29 and DLD-1 CRC cell lines. Expression of 15-LOX-1 protein was measured by immunoblot. Induction of apoptosis was evaluated by annexin V staining. All data are presented as mean ± SEM, with significance defined as P < 0.05. 15-LOX-1 in human CRC: From February 1998 to January 2002, 126 patients underwent surgical resection of either colorectal adenomas (n = 24) or carcinomas (n = 102), or both (n = 25). Tissue was macrodissected, snap frozen, and stored at −80°C. After tissue processing, RNA was extracted and gene expression of 15-LOX-1 was quantified utilizing ABI prism real-time quantitative RT-PCR. Significance evaluated by the Wilcoxon signed rank test. Results:Effect of celecoxib in cell culture: After 72 hours of treatment with celecoxib, immunoblot demonstrated a 1.5- to 2-fold increase in15-LOX-1 protein expression in HT-29 and DLD-1 cells, respectively. Celecoxib produced greater than a 2-fold increase in the rate of apoptosis compared with control cells in both cell lines (P < 0.05). 15-LOX-1 in human CRC: The mean age of the patients was 62 ± 1 years; 78% were white and 48% were female. The mean size of the polyps and cancers were 3.0 ± 0.4 and 5.0 ± 0.1 cm, respectively. Expression of 15-LOX-1 relative to S9 was 30 in normal mucosa and significantly down-regulated to 11 in adenomas and 16 in carcinomas (P < 0.05). Conclusions:15-LOX-1 gene expression is significantly reduced in both human colorectal adenomas and carcinomas and associated with decreased survival. Administration of celecoxib restores 15-LOX-1 protein expression and induces apoptosis. Down-regulation of 15-LOX-1 is an early event in the adenoma to carcinoma sequence, and reversal with celecoxib may represent one mechanism for chemoprevention of polyps or treatment of carcinomas.

ErbB3 expression promotes tumorigenesis in pancreatic adenocarcinoma

Historically, ErbB3 has been overlooked within the ErbB receptor family due to its perceived lack of tyrosine kinase activity. We have previously demonstrated that in pancreatic cancer ErbB3 is the preferred dimerization partner of EGFR, ErbB3 protein expression level directly correlates with the anti-proliferative effect of erlotinib (an EGFR-specific tyrosine kinase inhibitor), and transient knockdown of ErbB3 expression results in acquired resistance to EGFR-targeted therapy. In this study, we develop a stable isogenic model of ErbB3 expression in an attempt to decipher ErbB3s true contribution to pancreatic cancer tumorigenesis and to examine how this receptor affects cellular sensitivity to EGFR-targeted therapy. Analysis of the EGFR-ErbB3 heterodimer demonstrates that ligand-induced PI3K-AKT signaling is limited to ErbB3-expressing cells and that this signaling cascade can be partially abrogated by inhibiting EGFR function with erlotinib. Using our model of exogenous ErbB3 expression we showed a direct relationship between ErbB3 protein levels and increased pancreatic cancer cell proliferation in vitro. In vivo, ErbB3+PANC-1 xenografts had a significantly larger tumor volume than PANC-1 control xenografts (ErbB3-PANC-1) and displayed increased sensitivity to EGFR-targeted therapy. In pancreatic cancer, ErbB3 appears to be critically involved in EGFR signaling as evidenced by its profound effect on cellular proliferation and its ability to influence response to EGFR-targeted therapy.

Pancreatic cancer epidermal growth factor receptor (EGFR) intron 1 polymorphism influences postoperative patient survival and in vitro erlotinib response.

BackgroundEpidermal growth factor receptor (EGFR) has a highly polymorphic CA repeat region that affects transcription efficiency and anti-EGFR drug sensitivity in carcinomas. Erlotinib is an EGFR tyrosine kinase inhibitor approved for pancreatic cancer treatment. We analyzed the impact of EGFR intron 1 CA repeat lengths in pancreatic cancer clinical outcome and in vitro response to erlotinib.MethodsAllele-specific EGFR intron 1 lengths were analyzed in 30 microdissected pancreatic cancer surgical specimens, matched peripheral blood samples, and 9 pancreatic cancer cell lines treated with erlotinib. CA repeat lengths were correlated with survival, tumor parameters, molecular markers of EGFR pathway activation, and in vitro antiproliferative effects of erlotinib.ResultsBoth patient samples and cell lines displayed the full spectrum of EGFR CA repeat lengths (14–22 per allele). Patients with shorter sum of total CA repeats (<36) had worse median survival than patients with ≥36 repeats (13.7 vs 30.6 months, P = .002). Shorter patient EGFR intron 1 length correlated with EGFR expression (P = .026). Tumor intron 1 length was identical to that of matched peripheral blood specimens. There was no correlation between EGFR intron 1 length and pancreatic cancer stage, nodal status, grade, or expression of p-EGFR, p-ERK and p-Akt. Shorter EGFR intron 1 length was associated with in vitro response to erlotinib treatment (P = .02).ConclusionsShorter EGFR intron 1 CA repeat length is associated with worse pancreatic cancer clinical prognosis and in vitro response to erlotinib. EGFR intron 1 length can be reliably measured in peripheral blood and may translate into a quantitative predictive marker of both pancreatic cancer aggressiveness and erlotinib sensitivity.

Epigenetic downregulation of the DNA repair gene MED1/MBD4 in colorectal and ovarian cancer.

MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated (≥ 50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p

Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells

Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (−/−) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (−/−) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (−/−) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

Epidermal growth factor receptor (EGFR) intron 1 polymorphism and clinical outcome in pancreatic adenocarcinoma

BACKGROUND Epidermal growth factor receptor (EGFR) intron 1 has a polymorphic region of CA repeats that is believed to be associated with increased EGFR expression, tumor aggressiveness, and worse survival in cancer patients. METHODS A large population of pancreatic adenocarcinoma patients was investigated to evaluate this polymorphism as a potential prognostic marker of clinical outcome. Deoxyribonucleic acid obtained from 50 resected pancreatic adenocarcinomas and from 85 diagnostic endoscopic ultrasound-guided fine-needle aspiration procedures corresponding to patients with unresectable tumors was included. The correlation between CA repeat length and EGFR messenger ribonucleic acid levels was also examined. RESULTS Analysis of the 135 patients revealed no correlation between EGFR intron 1 CA repeat length and tumor stage. There was no difference in overall patient survival when stratified by allele length. A correlation between EGFR intron 1 length and EGFR transcript and protein levels could not be established. CONCLUSIONS The length of the EGFR intron 1 CA repeats does not correlate with levels of EGFR expression and cannot be used as marker of clinical prognosis in pancreatic cancer patients.

Abstract 2446: The effects of dacomitinib (PF-299) on pancreatic ductal adenocarcinoma (PDAC) and cancer-associated fibroblasts (CAFs).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: PF-299 is an oral irreversible small molecule that is an effective inhibitor of the ErbB pathway, which has been shown to play a critical role in pancreatic ductal adenocarcinoma (PDAC) pathogenesis. Indirect activation of Epidermal Growth Factor Receptor (EGFR) signaling through ErbB3 heterodimerization and stromal ligand production has been shown to act as an escape mechanism for EGFR directed therapies. This phenomenon may explain the modest clinical benefit of anti-EGFR drugs in PDAC. We have previously shown that CAFs are responsible for the secretion of ErbB pathway ligands. The addition of CAFs to PDAC xenografts improves the tumor modeling and adds testing stringency to novel targeted therapies. We hypothesize that PF-299 inhibition of multiple ErbB receptors results in effective inhibition of PDAC tumor progression. Materials: AsPC-1, BxPC-3, C3, Panc-1 and Panc-1+ErbB3 (stable ErbB3 transfected cell line) PDAC cells were treated with different concentrations of PF-299. PDAC cell proliferation and ErbB signaling was analyzed in vitro. Subcutaneous xenografts were developed using the AsPC-1 and BxPC-3 cell lines alone and in combination with CAFs. CAFs were obtained from surgically resected PDAC tissue. Xenografts were analyzed for tumor growth and tumor-associated ErbB signaling after treatment with PF-299. Results: In vitro, PF-299 effectively inhibited AsPC-1, BxPC-3, C3, Panc-1 and Panc-1+ErbB3 cell proliferation in a dose-dependent manner (1-10 μM). PF-299 decreased migration of AsPC-1 cells by 54%, while the addition of CAFs (in co-culture) increased cell migration by 13% in the PF-299 treated group compared to an increase of 273% in the control group. In vivo, tumor proliferation of AsPC-1 and BxPC-3 xenografts was significantly inhibited by PF-299 treatment (p=0.04 and p=0.002, respectively). Combination of AsPC-1/BxPC-3+CAFs xenografts increased initial tumor size by 22.5% (p=0.17) for the AsPC-1 cell line and by 11.6% (p=0.46) for the BxPC-3 cell line compared to cells without CAFs. The AsPC-1+CAFs PF-299 treated tumors were 4.1% smaller than placebo treated tumors (p=0.99), while BxPC-3+CAFs PF-299 treated tumor were 26% smaller than the placebo treated group (p=0.05). Immunoblot analysis of AsPC-1/BxPC-3+CAFs xenografts confirmed PF-299 mediated inhibition of both EGFR and ErbB3 mediated signaling. Conclusion: We demonstrated that PF-299 targets multiple ErbB receptors, including ErbB3. PF-299 significantly inhibited PDAC cell proliferation, migration and tumor progression by targeting EGFR and ErbB3 signaling. CAFs may provide a novel addition to current models by increasing tumor virulence while pan-ErbB receptor targeting may improve therapeutic approaches to PDAC. Citation Format: Brett L. Broussard, Alevtina Mikhaylina, Juan P. Arnoletti, Martin J. Heslin, Andrey Frolov. The effects of dacomitinib (PF-299) on pancreatic ductal adenocarcinoma (PDAC) and cancer-associated fibroblasts (CAFs). [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 2446. doi:10.1158/1538-7445.AM2013-2446

Abstract 1171: Gastrointestinal stromal tumors (GIST) cell proliferation inhibition with combined adenoviral and molecular targeted therapy

Introduction: GIST, the most common mesenchymal malignancies of the GI tract, are characterized by gain-of-function mutations in KIT and PDGFRα receptors. Imatinib and sunitinib are tyrosine kinase inhibitors (TKI) currently approved for the treatment of GIST. Recent evidence suggests that GIST cells acquire secondary resistance to TKI through activation of alternative signaling pathways. We hypothesize that gene therapy with replicative vector oncolysis (virotherapy) can be utilized to improve efficacy of TKI and overcome GIST resistance to treatment. Objectives: To determine if a panel of oncolytic adenoviruses in combination with imatinib and sunitinib, results in synergistic inhibition of GIST cell proliferation. Results: GIST882 and GIST-T1 cells were subjected to imatinib and sunitinib treatment in combination with a panel of oncolytic adenoviruses (Ad5, Ad5RGD, Ad5pK7, and Ad5 - Ad3 fiber knob), to assess their inhibitory effects on cell proliferation. The Ad5/3 vector had the best rate of gene transfer and cytolytic effect. The greatest anti-proliferative effects were observed with 100 viral particles/cell of the Ad5/3 vector combined with TKI concentrations of 0.1 µM in GIST-T1 cell line. The combination of TKI and oncolytic adenoviruses showed greater inhibition of GIST cell proliferation (78% decrease) when compared to TKI alone (58% decrease) and viral therapy alone (23% decrease, p Conclusions: The oncolytic adenovirus Ad5/3 infects and inhibits GIST cell proliferation both in vitro and ex vivo. Virotherapy with Ad5/3 has synergistic anti-proliferative effects with TKI and may help overcome GIST resistance to KIT targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1171.