Venkateshwar Madka

The Epidermal Growth Factor Receptor Inhibitor Gefitinib Prevents the Progression of Pancreatic Lesions to Carcinoma in a Conditional LSL-KrasG12D/+ Transgenic Mouse Model

Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy with a dismal prognosis. Developing novel strategies to prevent or delay pancreatic cancer is currently of intense interest. The chemopreventive efficacy of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, was evaluated against the progression of pancreatic intraepithelial neoplasms (PanIN) to PDAC in conditional LSL-KrasG12D/+ transgenic mice. LSL-KrasG12D/+ and p48Cre/+ mice were bred, and offspring of activated KrasG12D/+ were generated. Six-week-old male KrasG12D/+ (20 per group) and C57BL/6 wild-type (12 per group) mice were fed (AIN-76A) diets containing 0, 100, and 200 ppm of gefitinib for 35 weeks. At termination, pancreases were evaluated histopathologically for PanINs and PDAC, and various biomarkers were measured by immunohistochemistry, immunofluorescence, immunoblotting, and/or reverse transcription-PCR. Dietary gefitinib at 100 and 200 ppm significantly suppressed PDAC incidence by 77% and 100%, respectively (P < 0.0001) when compared with control diet. Importantly, a significant inhibition of carcinoma and a dose-dependent suppression of PanINs [PanIN-1, 37-62% (P < 0.002); PanIN-2, 38-41 (P < 0.001); and PanIN-3, 7-34% (P < 0.0141)] were observed in mice treated with gefitinib. Furthermore, mice treated with 100 and 200 ppm of gefitinib exhibited 67.6% to 77.3% of the pancreas to be free from ductal lesions. Also, gefitinib reduced EGFR, proliferating cell nuclear antigen, cyclin D1, C2GNT, RhoA, β-catenin, p38, phospho-extracellular signal–regulated kinase, caveolin-1, and mucin and increased cyclin B1 in the pancreatic lesions/PDAC. In summary, these results show that gefitinib can prevent the progression of pancreatic cancer precursor lesions to PDAC in a preclinical model. The present study highlights the promise of chemoprevention and the potential usefulness of EGFR inhibitors in individuals at high risk for pancreatic cancer. Cancer Prev Res; 3(11); 1417–26. ©2010 AACR.

Anti-inflammatory phytochemicals for chemoprevention of colon cancer.

Every year more than a million new cancer cases and 600,000 deaths are reported world-wide. Colorectal cancer is the fourth most commonly occurring and second leading cause of cancer deaths in the United States. Significant progress has been made in understanding colorectal cancer through epidemiological, laboratory and clinical studies. Development of metastatic adenocarcinomas is a multistage process occurring over several years during which multiple genetic alterations and pathophysiological changes are associated. Colorectal cancer can be prevented if the transformation of normal colonic crypt cells to malignant can be halted or reversed. Some of the key molecules that are altered significantly and play important roles in colorectal tumor progression are associated with inflammation. Since chronic inflammation is now recognized as a potential risk factor for tumor development, targeting inflammatory pathways has proven effective in preventing formation of colonic tumors and their malignant progression in both preclinical and clinical studies. Synthetic non-steroidal anti-inflammatory drugs (NSAIDS) have been identified as potential colorectal cancer chemopreventive agents; however, most of these synthetic agents are associated with unwanted and sometimes fatal side effects. There is mounting evidence in support of the efficacy of naturally-occurring phytochemicals possessing anti-inflammatory activity. In this review we discuss key inflammatory pathways associated with colorectal cancer and promising naturally-occurring phytochemicals as anti-inflammatory agents for the prevention and treatment of colorectal cancer.

Simultaneous targeting of 5-LOX-COX and EGFR blocks progression of pancreatic ductal adenocarcinoma

Cyclooxygenase-2 (COX-2), 5-Lipoxygenase (5-LOX), and epidermal growth factor receptor (EGRF) are over-expressed in human pancreatic ductal adenocarcinoma (PDAC). Using next-generation sequencing (NGS) analysis, we show significant increase in COX-2, 5-LOX, and EGFR expression during PDAC progression. Targeting complementary pathways will achieve better treatment efficacy than a single agent high-dose strategy that could increase risk of side effects and tumor resistance. To target COX-2, 5-LOX, and EGFR simultaneously, we tested effects of licofelone (dual 5-LOX-COX inhibitor), and gefitinib (EGFR inhibitor), individually and in combination, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PDAC using genetically engineered mice. Individually, licofelone (L) and gefitinib (G) significantly inhibited incidence of PDAC in male (72% L, 90% G, p < 0.0001) and female (90% L, 85% G, p < 0.0001) mice. The combination drug treatment produced complete inhibition of PDAC in both genders. Pancreata of mice receiving combination treatment showed significantly fewer Dclk1-positive cancer stem-like cells, inhibition of COX-2, 5-LOX, PCNA, EGFR and β-catenin expression (p < 0.05–0.0002), increased p21 expression. Significant changes in tumor immune responses and desmoplastic reaction was observed by NGS analysis in combination treatment (p < 0.05). In summary, early simultaneous targeting of 5-LOX-COX- and EGFR pathways may provide additive inhibitory effects leading to complete suppression of PDAC.

Evaluation of 99mTc-Probestin SPECT As a Novel Technique for Noninvasive Imaging of Kidney Aminopeptidase N Expression

Aminopeptidase N (APN; CD13; EC 3.4.11.2) is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 h postinjection of 99mTc-probestin in a group of 12 UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrate the potential use of 99mTc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.

Current Challenges and Opportunities for Chemoprevention of Pancreatic Cancer

BACKGROUND The incidence of pancreatic cancer (PC) is rising in parallel with the deaths caused by this malignant disease largely due to limited improvement in current treatment strategies. In spite of aggressive PC research, for the past three decades, there has been no significant improvement in the five-year survival for this cancer. Like many other cancers, it takes several years for normal pancreatic cells to transform into pancreatic precursor lesions and to further progress into invasive carcinoma. Hence there is a scope for the development of chemo-preventive strategies to inhibit/ delay/prevent progression of this disease into an advanced stage cancer. OBJECTIVE Chemoprevention of pancreatic cancer. METHOD Review of published literature. RESULTS AND CONCLUSION Availability of various genetically engineered mouse (GEM) models of PC has led to accelerated progress in understanding the disease and developing intervention strategies otherwise stalled for a long time. These GEM models spontaneously develop PC in a stepwise manner and mimic the disease etiology in humans. Understanding PC development from initiation to progression to metastasis is very important for early detection and prevention of PC. In this review, we focus on the current situation, the potential challenges, the progress in existing strategies and available opportunities as well as suggest key areas for research within the increasingly important area of pancreatic cancer chemoprevention.

Clinically Relevant Anti-Inflammatory Agents for Chemoprevention of Colorectal Cancer: New Perspectives

Substantial efforts are underway for prevention of early stages or recurrence of colorectal cancers (CRC) or new polyp formation by chemoprevention strategies. Several epidemiological, clinical and preclinical studies to date have supported the chemopreventive potentials of several targeted drug classes including non-steroidal anti-inflammatory drugs (NSAIDs) (aspirin, naproxen, sulindac, celecoxib, and licofelone), statins and other natural agents—both individually, and in combinations. Most preclinical trials although were efficacious, only few agents entered clinical trials and have been proven to be potential chemopreventive agents for colon cancer. However, there are limitations for these agents that hinder their approval by the food and drug administration for chemoprevention use in high-risk individuals and in patients with early stages of CRC. In this review, we update the recent advancement in pre-clinical and clinical development of selected anti-inflammatory agents (aspirin, naproxen, sulindac, celecoxib, and licofelone) and their combinations for further development as novel colon cancer chemopreventive drugs. We provide further new perspectives from this old research, and insights into precision medicine strategies to overcome unwanted side-effects and overcoming strategies for colon cancer chemoprevention.

Abstract 4984: Celastrol inhibits high fat diet-induced obesity and intestinal tumorigenesis in APCMin/+mice by modulating gut microbes and inflammation

Obesity and inflammation play a vital role in colorectal cancer (CRC). Antiobesity agents may be beneficial for CRC prevention. Celastrol is a triterpene bioactive compound derived from Tripterygium wilfordii (TW) plant, which possesses antiobesity and anti-inflammatory properties. In the present study, we tested celastrol for its intestinal tumor inhibitory efficacy, modulation of intestinal microbiome, induction of UCP-1 in inguinal fat, and inflammation under obese conditions using APCMin/+ mouse model. For efficacy study, six-week-old male and female C57BL/6J-APCMin/+ mice (10 mice/group/gender) were fed high-fat diets (HFD; 60% Kcal fat) containing 0 and 150 ppm celastrol and a group of mice with a low-fat diet (LFD; 10% Kca fat) for 11 weeks. At termination, intestinal tumors were evaluated histologically and serum was assayed for fasting glucose, uric acid, liver enzymes (ALT, AST), triglycerides and cholesterol levels. Untreated and treated intestinal tumors were assayed for apoptosis and inflammatory markers by real-time PCR method. Results suggest that administration of LFD showed lower intestinal tumor formation by 52% (p Stephenson Cancer Center Grant. Citation Format: Naveena B. Janakiram, Venkateshwar Madka, Yuting Zhang, Nicole Stratton, Hima Bezawada, Beth Griesel, Altaf Mohammed, Ann L. Olson, Chinthalapally V. Rao. Celastrol inhibits high fat diet-induced obesity and intestinal tumorigenesis in APCMin/+ mice by modulating gut microbes and inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4984.

Lack of chemopreventive effects of P2X7R inhibitors against pancreatic cancer

Pancreatic cancer (PC) is an almost uniformly lethal disease with inflammation playing an important role in its progression. Sustained stimulation of purinergic receptor P2X7 drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome, we performed transcriptomic analysis of p48Cre/+-LSL-KrasG12D/+ mice pancreatic tumors by next generation sequencing. Results showed that P2X7Rs key inflammasome components, IL-1β and caspase-1 are highly expressed (p < 0.05) in pancreatic tumors. Hence, to target P2X7R, we tested effects of two P2X7R antagonists, A438079 and AZ10606120, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PC in p48Cre/+-LSL-KrasG12D/+ mice. Following dose optimization studies, for chemoprevention efficacy, six-week-old p48Cre/+-LSL-KrasG12D/+ mice (24–36/group) were fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC incidence. A marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug-treated mice. Importantly, the carcinoma spread in untreated mice was 24% compared to 43–53% in treatment groups. Reduced survival rates were observed in mice exposed to P2X7R inhibitors. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of P2X7R, NLRP3, and IL-33, whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show chemopreventive effects against PC and slightly enhanced PanIN progression to PDAC. Hence, caution is needed while treating high-risk individuals with P2X7R inhibitors.

Targeting Inflammation for Bladder Cancer Chemoprevention

Purpose of ReviewCancer of the bladder is a serious health problem with significant mortality once it progresses to advanced stages. Moreover, chronic surveillance and treatments are required to prevent the inherent high recurrence and aggressive nature of these tumors. As a result, not only the quality of life for the patients is affected but it also adds to the treatment cost, making it one of the costliest cancers to treat. Therefore, it is highly imperative to consider preventive options that have been largely neglected.Recent FindingsAlthough smoking prevalence is decreasing, a decline in BC incidence has not been seen, yet suggesting that smoking history and other factors still pose a threat to develop BC. With the increase of diabetic and obese populations, the risk for BC is also increasing. In spite of their modifiable nature, advancement in diagnosis, understanding of the disease, and enormous preclinical chemoprevention data, efforts in that direction for screening and primary or secondary prevention of this disease have been unsatisfactory. Moreover, no new therapeutic were approved for the last three decades; thus, the 5-year survival of BC patients has also not improved for decades.SummaryIn the current review, we have discussed the central role of inflammation in the major risk factors such as smoking, diabetes, obesity, and infection leading to BC. We summarized the preclinical data of promising anti-inflammatory agents for primary, secondary, as well as tertiary prevention. Thus, we believe that developing chemopreventive strategies for bladder cancer by targeting inflammation is highly desirable.

Abstract 5260: NGS Transcriptome analysis of colon adenocarcinomas treated with individual and combination of aspirin and omeprazole

Colorectal cancer (CRC) is the third most common type of cancer in men and women in US. Anti-inflammatory agents have proven to be most effective in CRC prevention, but are associated with gastrointestinal toxicities. Hence, to reduce the acidic environment and enhance the chemopreventive efficacy of anti-inflammatory agent aspirin, a combination of aspirin with acidity neutralizer omeprazole was used in AOM-induced F344 Rat model of CRC. Rat (36/group) colon cancers were induced by AOM (15mg/Kg body weight) s.c., once a week for two weeks. At adenoma stage (13 wks of age), rats were fed diets containing aspirin (700 or 1400ppm), omeprazole (0, 250 or 500 ppm), or aspirin + omeprazole (700 ppm + 250 ppm; 1,400 ppm + 500 ppm). To identify a molecular predictor of the benefit of aspirin plus omeprazole we performed next-generation sequencing of colon adenocarcinomas (AdCa) treated with aspirin (1400 ppm), omeprazole (500 ppm), aspirin plus omeprazole (1400 plus 500 ppm),conducted whole-transcriptome analysis and correlated the molecular portrait with chemoprevention benefit. Aspirin plus omeprazole enhanced colon AdCa incidence inhibitory effects by 55% (p Citation Format: Naveena B. Janakiram, Altaf Mohammed, Jagan M.R. Patlolla, Yuting Zhang, Laura Biddick, Venkateshwar Madka, Li Qian, Stan Lightfoot, Barbara Dunn, Ronald Lubet, Chen S. Suen, Vernon E. Steele, Chinthalapally V. Rao. NGS Transcriptome analysis of colon adenocarcinomas treated with individual and combination of aspirin and omeprazole [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5260. doi:10.1158/1538-7445.AM2017-5260