Bassel F. El-Rayes

Exploitation of protein kinase C : A useful target for cancer therapy

Protein kinase C is a family of serine/threonine kinases. The PKC family is made up of at least 12 isozymes, which have a role in cell proliferation, differentiation, angiogenesis, and apoptosis. Activation of PKC isozyme is dependent on tyrosine-kinase receptors and G-protein-coupled receptors. PKC isozymes regulate multiple signaling pathways including PI3-K/Akt, MAPK, and GSK-3beta. PKC isozymes have variable roles in tumor biology which in part depend on the cell type and intracellular localization. PKC isozymes are commonly dysregulated in the cancer of the prostate, breast, colon, pancreatic, liver, and kidney. Currently, several classes of PKC inhibitors are being evaluated in clinical trials and several challenges in targeting PKC isozymes have been recently identified. In conclusion, PKC remains a promising target for cancer prevention and therapy.

Apoptosis-inducing effect of erlotinib is potentiated by 3,3′-diindolylmethane in vitro and in vivo using an orthotopic model of pancreatic cancer

Blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase inhibitors is insufficient for effective antitumor activity because of independently activated survival pathways. A multitargeted approach may therefore improve the outcome of anti-EGFR therapies. In the present study, we determined the effects of 3,3′-diindolylmethane (Bioresponse BR-DIM referred to as B-DIM), a formulated DIM with greater bioavailability on cell viability and apoptosis with erlotinib in vitro and in vivo using an orthotopic animal tumor model. BxPC-3 and MIAPaCa cells with varying levels of EGFR and nuclear factor-κB (NF-κB) DNA-binding activity were treated with B-DIM (20 μmol/L), erlotinib (2 μmol/L), and the combination. Cell survival and apoptosis was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and histone-DNA ELISA. Electrophoretic mobility shift assay was used to evaluate NF-κB DNA-binding activity. We found significant reduction in cell viability by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, induction of apoptosis, down-regulation of EGFR phosphorylation, NF-κB DNA-binding activity, and expression of antiapoptotic genes in BxPC-3 cells when treated with the combination of erlotinib and B-DIM compared with either agent alone. In contrast, no such effect was observed in MIAPaCa cells by similar treatment. Most importantly, these in vitro results were recapitulated in animal model showing that B-DIM in combination with erlotinib was much more effective as an antitumor agent compared with either agent alone. These results suggest that the utilization of B-DIM could be a useful strategy for achieving better treatment outcome in patients with activated status of EGFR and NF-κB in their tumors. [Mol Cancer Ther 2008;7(6):1708–19]

Protein kinase C: A target for therapy in pancreatic cancer

Objectives: Protein kinase C (PKC) is involved in tumor growth and apoptosis and hence represents a potential target for cancer therapy. This study investigated the expression of PKC in pancreatic tumor tissue in comparison to adjacent normal tissue and determined the modulation of PKC by bryostatin-1 (BRYO) on pancreatic cancer cell lines. Methods: Pancreatic tissue was obtained from 18 patients who had a resection (14 with ductal adenocarcinoma and 4 with adenoma and high-grade dysplasia). Cytosolic and nuclear membrane PKCs in the paired samples were determined by immunoblotting. HPAC cells were treated with gemcitabine and BRYO and in sequential and concomitant combination. To evaluate cell viability, apoptosis, and electrophoretic mobility shift assay, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, enzyme-linked immunosorbent assay, and nuclear factor &kgr;B (NF-&kgr;B) assays were used. Results: As compared with the adjacent normal tissue, PKC-&agr;, PKC-&bgr;1, and PKC-&dgr; were higher in the tumor; PKC-ϵ was higher in the normal tissue. Pretreatment with gemcitabine followed by BRYO resulted in decreased cell viability, increased apoptosis, and inhibited NF-&kgr;B than either agent alone or BRYO followed by gemcitabine. Conclusion: Protein kinase C is overexpressed and activated in pancreatic cancer as compared with normal tissue. Inhibition of PKC could sensitize pancreatic cancer cell lines to the effects of gemcitabine. The potentiation of gemcitabine by BRYO is sequence-dependent and mediated through inhibition of PKC-dependent activation of NF-&kgr;B.

Concurrent Inhibition of NF-κB, Cyclooxygenase-2, and Epidermal Growth Factor Receptor Leads to Greater Anti-Tumor Activity in Pancreatic Cancer

Inactivation of survival pathways such as NF‐κB, cyclooxygenase (COX‐2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3′‐Diindolylmethane (B‐DIM) is an inhibitor of NF‐κB and COX‐2 and is a well‐known chemopreventive agent. We hypothesized that the inhibition of NF‐κB and COX‐2 by B‐DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti‐tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B‐DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3‐(4,5‐dimetylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX‐2, EGFR, and NF‐κB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down‐regulation in the expression of COX‐2, NF‐κB, and EGFR in BxPC‐3, COLO‐357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF‐κB, and COX‐2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B‐DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi‐targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX‐2, EGFR, and NF‐κB. J. Cell. Biochem. 110: 171–181, 2010.

Squamous cell cancer of the anal canal in HIV-infected patients receiving highly active antiretroviral therapy: a single institution experience.

Background:Human immunodeficiency virus (HIV)-infected patients are at increased risk for squamous cell carcinoma of the anal canal (SCCA) and the incidence of SCCA has increased in the era of highly active antiretroviral therapy. The outcome of SCCA in HIV-positive patients has not been evaluated in prospective trials and the published literature is limited to retrospective case series. The aim of this study is to describe the treatment, toxicity, and overall survival (OS) in patients with and without HIV infection. Methods:We performed a retrospective chart review of all patients treated for invasive SCCA at Karmanos Cancer Institute, Wayne State University from 1991 to 2007 and collected data regarding HIV status, demographics, stage at diagnosis, treatment, response to treatment, toxicity, and survival. Results:Forty-five patients with SCCA were identified, of whom 13 were HIV-positive and 32 were HIV-negative. HIV-positive patients were younger (median age, 45 vs. 57 years) and had a higher frequency of men (89% vs. 37%). Patients were balanced for presenting stage at diagnosis and rates of local recurrence were found to be similar between the 2 groups. HIV-positive patients were less likely to receive full dose chemoradiotherapy. Except for dermatitis, the incidence of grade 3 to 4 toxicities was similar in both groups. Median OS was 33.5 months for HIV-positive patients and 71.8 months for HIV-negative patients. Although limited by the small size of the study, the OS was not statistically significantly different by HIV status (P = 0.787). Conclusion:Although the HIV-positive patients received lower dose chemoradiotherapy, no major difference in local control or overall survival was observed.

CA19‐9 as a predictor of tumor response and survival in patients with advanced pancreatic cancer treated with gemcitabine based chemotherapy

Aims:  The aim of this study was to determine the predictive role of pretreatment carbohydrate antigen 19‐9 (CA19‐9) measurement and its change after one cycle of gemcitabine‐based therapy for response, time to progression (TTP) and overall survival (OS).

Protein kinases C isozymes are differentially expressed in human breast carcinomas

AIMS The protein kinase C (PKC) family of enzymes has been implicated in cellular proliferation, differentiation, and apoptosis. However, the distribution of specific PKC isoforms with varying functions in normal and malignant human tissues remains to be determined. The objective of this study was to investigate the expression of certain PKC isoforms (alpha, betaI, betaII, epsilon) in human breast cancer specimens relative to adjacent uninvolved tissue (n=24) and in the normal breast tissue obtained from patients undergoing reduction mammoplasty (n=12). MAIN METHODS Western blot analysis using PKC isoform specific antibodies was performed on tissue extracts from breast tumors, adjacent uninvolved tissues, and reduction mammoplasty tissues. KEY FINDINGS Mean levels of cytosolic and membrane PKC-alpha, PKC-betaI, and PKC-betaII were significantly higher in the cancer specimens than in the adjacent uninvolved breast tissues (Wilcoxon signed-ranks test; P<0.05 for each, after adjustment for multiple comparisons). There was a notably higher mean level of membrane PKC-betaII isozyme in Her-2 positive and in poorly differentiated tumors. No significant differences were observed when normal tissue adjacent to tumor was compared to breast tissue obtained from reduction mammoplasty specimens. SIGNIFICANCE Higher level of PKC-alpha, PKC-betaI, and PKC-betaII in cancer specimens and higher level of PKC-betaII in Her-2 positive tumors require further exploration of the intracellular pathways involving PKC-alpha and -beta isoforms in breast cancer because both could be specific targets for the development of new therapies and for the prevention and treatment of this disease.

Impact of race, age, and socioeconomic status on participation in pancreatic cancer clinical trials

Objectives: Over 18 years, 7 phase 2 trials in advanced pancreatic cancer (APC) were conducted at Karmanos Cancer Institute (KCI). We sought factors that influenced the selection of patients for clinical trials and explored differences in overall survival (OS) of patients treated on clinical trials versus standard of care. Methods: The target population was patients with APC diagnosed between January 1, 1986, and December 31, 2003. Patients were divided into 3 mutually exclusive groups: treated on clinical trials at KCI (t-KCI), treated at KCI but not on a clinical trial (KCI), or treated at non-KCI institutions (n-KCI). Results: Eight thousand two hundred thirty patients met study criteria: 6470 n-KCI, 1642 KCI, and 118 t-KCI. Significant differences were observed across the 3 groups with respect to age, race, stage, grade, and socioeconomic status. Median OS was higher in t-KCI (8.5 months) than in KCI (5.0 months) or n-KCI (2.8 months) and could not be accounted for by variations in baseline characteristics. Conclusions: Patients enrolled on clinical trials were younger, had better socioeconomic status, and were less often African American. Patients with APC treated at academic institutions may have longer OS than patients treated in the community. Clinical trials seem to offer a survival advantage for patients with APC.

Small molecule tyrosine kinase inhibitors in pancreatic cancer

Pancreatic cancer has proven to be chemo-resistant, with gemcitabine being the only cytotoxic agent approved for advanced pancreatic cancer since 1996. Tyrosine kinase inhibitors represent a newer generation of chemotherapeutic agents targeting specific tumor pathways associated with carcinogenesis including cell cycle control, signal transduction, apoptosis and angiogenesis. These agents present a more selective way of treating pancreatic cancer. Erlotinib is the prototype of the tyrosine kinase inhibitors with proven efficacy in advanced pancreatic cancer and has been recently approved in that setting. Multiple other tyrosine kinase inhibitors targeting the VEGFR, PDGFR, and Src kinases are in various phases of clinical trials testing. The preliminary results of these trials have been disappointing. Current challenges in pancreatic cancer clinical trials testing include improving patient selection, identifying effective combinations, improving the predictive value of current preclinical models and better study designs. This review summarizes the present clinical development of tyrosine kinase inhibitors in pancreatic cancer and strategies for future drug development.

An Overview of Clinical Trials of Targeted Therapies in Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of cancer mortality in the United States ( 1 ). Over 75% of patients with pancreatic cancer have locally advanced or metastatic disease at diagnosis ( 2 ). The median survival of patients with metastatic and locally advanced pancreatic cancer is 3–6 months and 8–10 months, respectively. The only potentially curative treatment is surgical resection. Patients who undergo a potentially curative resection have a median survival of 14–20 months and <20% are long-term survivors because of the development of metastatic disease. Therefore, improvement in the outcome of patients with pancreatic cancer is dependent on the development of more effective systemic therapies. Gemcitabine is considered the standard chemotherapy in advanced pancreatic cancer. The response rate, median survival and 1-year survival in patients with advanced pancreatic cancer treated with gemcitabine in the pivotal trial were 5.4%, 5.6 months, and 18%, respectively ( 3 ). Combination chemotherapy did not demonstrate any significant survival advantage in randomized trials as compared with single-agent gemcitabine ( 4 , 5). In the adjuvant setting, gemcitabine has been shown to be superior to observation ( 6) or 5-fluorouracil ( 7 ). Therefore, the impact of cytotoxic chemotherapy in pancreatic cancer has been at best modest. Nevertheless, most of the new drug development using targeted agents has been based on a gemcitabine platform. Fundamental processes associated with carcinogenesis include molecular aberrations involving cell cycle control, signal transduction, apoptosis, angiogenesis, and invasion. The identification of agents that target these abnormalities offers an opportunity for the development of effective and selective systemic treatments for pancreatic cancer. Unfortunately, recently conducted clinical trials evaluating the role of novel targeted therapies in pancreatic cancer have demonstrated somewhat disappointing results (Table 33.1 ). This chapter reviews the clinical studies that have been undertaken in patients with pancreatic cancer using targeted agents.