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Purpose: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed. Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance. Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib (P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy. Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET.

In many areas of oncology, we lack sensitive tumor-burden monitoring to guide critical decision making. While circulating tumor DNA (ctDNA) promises to enable disease monitoring, this approach is limited by the sparsity of ctDNA in the plasma. To overcome this challenge, error-corrected deep targeted sequencing has been proposed. Nonetheless, this framework is limited by the low number of genomic equivalents (GEs, ~103/mL of plasma), imposing a ceiling on effective sequencing depth. We have previously shown that genome-wide mutational integration through plasma whole genome sequencing (WGS) can sever the dependency between available GEs and assay sensitivity (Zviran et al, 2020). In this approach, tumor-informed mutational profiles are applied to plasma WGS, allowing detection of tumor fractions as low as 10−5. However, the higher cost of WGS limits practical depth of coverage (20-30X) and may limit broad adoption. Lower costs may thus allow for enhanced ctDNA cancer monitoring via WGS. We therefore applied emerging lower-cost WGS (1USD/Gb, Almogy et al, 2022) to plasma from 7 patients with metastatic cancer at ~115x coverage depth. Read depth profiling and error rates were comparable between matched Ultima and standard platform datasets. Integration of deep learning architectures for signal to noise enrichment (Widman et al, biorxiv, 2022) with deeper WGS coverage enabled ctDNA detection at the parts per million range. We reasoned that lower sequencing cost can be harnessed for duplex error-corrected WGS. Proof-of-concept experiments in mouse PDX samples showed ~1,500x decrease in errors. Applied to the plasma of stage IV melanoma patients (n=5), we obtained error rates ~10−7. We used this approach to tackle the challenging context of cancer monitoring in early-stage melanoma without matched tumor sequencing. While in uncorrected WGS, de novo mutation calling yielded limited ability to detect melanoma specific mutations, duplex-corrected WGS allowed us to harness melanoma mutational signatures for disease monitoring without matched tumor profiling. Analytic validation of our assay showed sensitive and specific cancer detection when the concentration of ctDNA was at 10−4 concentrations. Applied to a cohort of stage III melanoma patients with negative ctDNA detection using previously described methods, we detected ctDNA in all cases (n=4), demonstrating enhanced sensitivity using duplex WGS. These data demonstrate the exciting potential of low cost WGS for ultra-sensitive ctDNA cancer monitoring. In the tumor-informed setting, deeper sequencing increased sensitivity for mutational profile detection. Moreover, the application of duplex error-correction at genome scale allowed for sensitive cancer monitoring without matched tumor profiles. We envision that the era of low-cost sequencing will empower ultra-sensitive cancer monitoring via WGS, with transformative impact on cancer care. Citation Format: Alexandre P. Cheng, Adam J. Widman, Anushri Arora, Itai Rusinek, William F. Hooper, Rebecca Murray, Daniel Halmos, Theophile Langanay, Giorgio Inghirami, Soren Germer, Melissa Marton, Adrienne Helland, Rob Furatero, Jaime McClintock, Lara Winterkorn, Zoe Steinsnyder, Yohyoh Wang, Srinivas Rajagopalan, Asrar I. Alimohamed, Murtaza S. Malbari, Ashish Saxena, Margaret K. Callahan, Dennie T. Frederick, Lavinia Spain, Ariel Jaimovich, Doron Lipson, Samra Turajlic, Michael C. Zody, Nasser K. Altorki, Jedd D. Wolchok, Michael A. Postow, Nicolas Robine, Genevieve Boland, Dan A. Landau. Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5709.

BACKGROUND Antibiotic use can result in reduced efficacy of immune checkpoint blockade (ICB), presumably because of dysbiosis of the intestinal microbiome. We sought to determine the precise temporal relation between antibiotic therapy and its possible effects on ICB efficacy. We also investigated the histologic changes in the tumor microenvironment secondary to antibiotics use. METHODS AND OBJECTIVES This was a single institution retrospective study that evaluated the impact of antibiotics on outcomes of patients with advanced or metastatic malignancy who were treated with ICB. Use of antibiotics among patients treated with ICB was assessed during a 12-week period before and after initiation of ICB. The primary outcome was response to ICB. Histologic changes in the tumor microenvironment following antibiotics use were also examined. RESULTS Between January 1, 2011 and December 31, 2018, 414 patients were identified who received ICB, and 207 patients (50%) received antibiotics within 12 weeks (before/after) of initiation of ICB. In univariate analysis, antibiotic use following initiation of ICB was associated with a significantly reduced response (odds ratio [OR]: 0.33, 95% confidence interval [CI]: 0.2-0.52, P

Lung cancer is the leading cause of cancer-related deaths in the United States. Identifying new factors that promote drug resistance is critical to restoring drug sensitivity and improving patient outcomes. Methylmalonic acid (MMA) is a dicarboxylic acid by-product of propionate metabolism. MMA levels rise in the serum with age and are associated with cancer-related mortality (the higher the MMA, the worse the mortality). We recently asked whether this metabolite had a functional impact and showed that treating non-small cell lung cancer (NSCLC) cells with MMA in vitro promotes drug resistance through triggering epithelial-to-mesenchymal transition (EMT). However, whether MMA is elevated in lung cancer is not known, and how MMA levels are regulated is poorly understood. To determine if MMA is elevated in NSCLC, we performed targeted metabolomics on 20 resected lung adenocarcinomas and squamous cell carcinomas with matched normal lung tissue. We found that MMA was 2.5 fold elevated in tumors compared to controls (p Citation Format: Bobak Parang, Zhongchi Li, Vivien Low, Jennifer Endress, Murtaza Malbari, Ashish Saxena, Nasser Altorki, John Blenis. Methylmalonic acid is elevated in non-small cell lung cancer and promotes drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2378.

PURPOSE The American Society for Radiation Oncology (ASTRO) produced an evidence-based guideline on radiation therapy (RT) for small-cell lung cancer (SCLC). Because of the relevance of this topic to ASCO membership, ASCO reviewed the guideline, applying a set of procedures and policies used to critically examine guidelines developed by other organizations. METHODS The ASTRO guideline on RT for SCLC was reviewed for developmental rigor by methodologists. Then, an ASCO Expert Panel reviewed the content and the recommendations. RESULTS The ASCO Expert Panel determined that the recommendations from ASTRO guideline on RT for SCLC, published in June 2020, are clear, thorough, and based upon the most relevant scientific evidence. ASCO endorsed ASTRO guideline on RT for SCLC with a few discussion points. RECOMMENDATIONS Recommendations addressed thoracic radiotherapy for limited-stage SCLC, role of stereotactic body radiotherapy in stage I or II node-negative SCLC, prophylactic cranial radiotherapy, and thoracic consolidation for extensive-stage SCLC. Additional information is available at www.asco.org/thoracic-cancer-guidelines .

Background: Recent epidemiological and experimental studies have shown that obesity is a major risk factor for Colorectal Cancer (CRC). Regular intake of high fat-containing diet can promote obesity and metabolic syndrome by increasing the insulin resistance and inflammatory response which contribute to carcinogenesis. Previously, we have shown that inhibition of polyol pathway enzyme aldose reductase (AR) prevents carcinogens- and inflammatory growth factorsinduced CRC. However, the effect of AR inhibition on a high-fat diet (HFD)-induced formation of intestinal polyps in Apc-deficient Min (multiple intestinal neoplasia; ApcMin/+) mice is not known. Methods: We examined the effect of AR inhibitor, fidarestat on the HFD-induced formation of preneoplastic intestinal polyps in ApcMin/+ mice which is an excellent model of colon cancer. Results: APCMin/+ mice fed for 12 weeks of HFD caused a significant increase in the formation of polyps in the small and large intestines and fidarestat given along with the HFD prevented the number of intestinal polyps. Fidarestat also decreased the size of the polyps in the intestines of HFDtreated APC Min mice. Further, the expression levels of beta-catenin, PCNA, PKC-β2, P-AKT, Pp65, COX-2, and iNOS in the small and large intestines of HFD-treated mice significantly increased, and AR inhibitor prevented it. Conclusion: Our results thus suggest that fidarestat could be used as a potential chemopreventive drug for intestinal cancers due to APC gene mutations.

Estrogen hormones protect against colorectal cancer (CRC) and a preventative role of estrogen receptor beta (ERβ) on CRC has been supported using full knockout animals. However, it is unclear through which cells or organ ERβ mediates this effect. To investigate the functional role of intestinal ERβ during colitis-associated CRC we used intestine-specific ERβ knockout mice treated with azoxymethane and dextran sodium sulfate, followed by ex vivo organoid culture to corroborate intrinsic effects. We explored genome-wide impact on TNFα signaling using human CRC cell lines and chromatin immunoprecipitation assay to mechanistically characterize the regulation of ERβ. Increased tumor formation in males and tumor size in females was noted upon intestine-specific ERβ knockout, accompanied by enhanced local expression of TNFα, deregulation of key NFκB targets, and increased colon ulceration. Unexpectedly, we noted especially strong effects in males. We corroborated that intestinal ERβ protects against TNFα-induced damage intrinsically, and characterized an underlying genome-wide signaling mechanism in CRC cell lines whereby ERβ binds to cis-regulatory chromatin areas of key NFκB regulators. Our results support a protective role of intestinal ERβ against colitis-associated CRC, proposing new therapeutic strategies. QC 20201009

// Trang Nguyen-Vu 1, * , Jun Wang 1, * , Fahmi Mesmar 1 , Srijita Mukhopadhyay 1 , Ashish Saxena 1 , Catherine W. McCollum 1 , Jan-Ake Gustafsson 1, 2 , Maria Bondesson 1, 3 , Cecilia Williams 1, 2, 4 1 Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX, USA 2 Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden 3 Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA 4 Science for Life Laboratory, School of Biotechnology, KTH The Royal Institute of Technology, Solna, Sweden * These authors have contributed equally to this work Correspondence to: Cecilia Williams, email: Cecilia.williams@scilifelab.se Keywords: colorectal cancer, PROX1, estrogen receptor, microRNA, metastasis Received: October 05, 2015 Accepted: May 25, 2016 Published: June 07, 2016 ABSTRACT Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ERβ) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ERβ represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ERβ and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ERβ upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3’UTR. Through the generation of intestine-specific ERβ knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo . Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo . In conclusion, ERβ in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3’UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ERβ-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

Estrogen has been shown to protect women against colorectal cancer (CRC). In this study, using tissue-specific knockout mice, we characterize the role for intestinal estrogen receptor beta (ERβ) and, for the first time, demonstrate a direct preventive role against colitis-induced colon tumors in both sexes. By inducing colitis and CRC using azoxymethane and dextran sodium sulfate in intestine-specific ERβ knockout mice, we observe an increase in colon ulceration in addition to an enhanced development of neoplasms, with increased number of tumors in males and increased tumor size in females. We identify a local increase in TNFα expression and deregulation of NFκB targets. We note that males exhibit especially strong effects following intestinal ERβ knockout. The protection by intestinal ERβ against TNFα-induced damage is corroborated using ex vivo organoid culture, where we observe that activation of ERβ protects against TNFα-induced epithelial cell damage. Further, we characterize an underlying mechanism of action, using CRC cell lines with re-introduced ERβ, where ERβ binds to cis-regulatory chromatin areas of key NFκB regulators BCL3, BIRC3 and ATF3, and thereby modulates TNFα-induced signaling. With these results we define a clear role for intestinal ERβ and demonstrate an underlying mechanism, strongly supporting the notion that selective activation of intestinal ERβ has potential to prevent CRC in both sexes. Citation Format: Linnea Hases, Rajitha Indukuri, Madeleine Birgersson, Trang Nguyen-Vu, Rodrigo Lozano, Ashish Saxena, Johan Hartman, Jonna Frasor, Jan-Åke Gustafsson, Pekka Katajisto, Amena Archer, Cecilia Williams. Intestinal estrogen receptor beta suppresses colon inflammation and tumorigenesis in both sexes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2081.

TPS3648 Background: Trophoblast cell surface antigen (Trop-2) is highly expressed in many epithelial cancers (non–small-cell lung cancer [NSCLC], endometrial cancer, urothelial carcinoma [UC], and triple-negative breast cancer [TNBC]) and has been linked to aggressive disease and poor prognosis. SG is a Trop-2–directed antibody drug conjugate containing SN-38 (active metabolite of irinotecan) with a 7.5:1 drug-to-antibody ratio and unique hydrolyzable linker that allows for extracellular bystander effect. The phase 1/2 IMMU-132-01 basket study reported clinical activity with SG in patients with multiple tumor types not selected for Trop-2 expression including NSCLC (objective response rate [ORR]: 17%), TNBC (ORR: 33%), and UC (ORR: 31%).1-3 Results from the overall safety population (N=420) from this study found that SG was tolerable, with a predictable and manageable safety profile, and low discontinuation rates due to AEs. Methods: To test a biomarker-enrichment strategy with Trop-2, the TROPiCS-03 (TROP-2 Investigations in Cancer with SG) study was initiated. TROPiCS-03 (NCT03964727) is a multi-cohort, open-label, phase 2 study in patients with metastatic solid tumors - presently NSCLC (adenocarcinoma and squamous cell carcinoma), head and neck squamous cell carcinoma, and endometrial cancer - selected based on elevated Trop-2 expression by a validated IHC assay. Patients receive SG (10 mg/kg IV, days 1 and 8 every 21 days) and continue treatment until lack of clinical benefit or unacceptable toxicity. The primary endpoint is objective response rate (local assessment) and additional endpoints include clinical benefit rate, duration of response, progression-free survival, and safety. Females or males ≥18 years old who are histologically documented to have locally advanced or metastatic (M1, stage 4) solid tumors of the above types are eligible. Patients must have ECOG 0 or 1 and adequate clinical laboratory results to be enrolled. All subjects will have progressed after prior platinum-based chemotherapy and programmed death-ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) directed therapy. Patients who have previously received topoisomerase I inhibitors and those with known active CNS metastases are excluded. Approximately 160 patients will be enrolled in the trial overall; enrollment in the NSCLC cohort is currently in progress. References: Heist RS et al. J Clin Oncol. 2017;35:2790-7, Bardia A et al., NEJM. 2019;380:741-51.,Tagawa ST et al., Oral presentation; ASCO-GU 2019, San Francisco, CA. Clinical trial information: NCT03964727 .

12090 Background: Malnutrition is an underrecognized predictor of inferior cancer related outcomes. Subjective global assessment (SGA), a brief validated survey for malnutrition, may predict increased CT toxicity. This phase II RCT was performed to validate SGA as a predictive tool for malnutrition and to evaluate the impact of MINT on CT associated toxicity. Methods: CT naive pts screened by SGA were assigned to well-nourished (SGA A) or malnourished (SGA B/C) cohorts. Both cohorts were followed for CT delivery, toxicity, quality of life (QOL) by FACT-G, biomarkers, radiology, and survival. SGA B/C pts, stratified by regimen/disease, were randomized 1:1 to MINT vs. usual care. The MINT cohort received weekly registered dietician counseling and symptom assessment over the 8-week study period. Percent standard and planned CT doses were calculated. Wilcoxon rank sum tests were used for differences between groups, log-rank tests for survival, and multivariable linear regression for adjusted comparisons. Results: 186 eligible pts were enrolled (94 SGA A, 92 SGA B/C). SGA A were younger (median age [range]; 63 [22, 89] vs. 70 [22, 91], p = 0.011) and more fit (ECOG 0-1; 96.8% vs. 72.8%, p < 0.001). Baseline QOL was higher for SGA A (median [range], 87 [34, 115]) vs SGA B/C (70 [31, 101], p < 0.001). SGA A was associated with higher CT delivery: median proportion of planned CT (1 [Q1 0.87, Q3 1] vs 0.94 [0.70, 1], p = 0.022) and standard CT (0.91 [0.72, 1] vs. 0.74 [0.57, 0.95] p < 0.001). Adjusted for age/ECOG, SGA A remained associated with > 80% of planned (OR 2.32, p = 0.05) and standard (OR 2.33, p = 0.04) CT. SGA B/C pts (n = 92) were randomized to MINT vs usual care: median nutrition encounters MINT 5.5 vs. usual care 0.5; we observed no differences in CT delivery: median proportion of planned CT (0.91 [0.69, 1] vs. 0.94 [0.74, 1], p = 0.84) and standard CT (0.75 [0.58, 0.96] vs 0.71 [0.52, 0.99], p = 0.59). SGA A was associated with a longer 12-month survival (77.8% [95% CI 69.6%, 86.9%]) vs. B/C (53.3% [42.8%, 66.4%], p < 0.0001; 12-month survival was similar for MINT (52.3% [38.1%, 71.9%]) vs usual care (54.4% [40.2%, 73.6%], p = 0.58). Conclusions: SGA is a validated tool to characterize malnutrition in pts receiving CT. Malnourished pts received significantly less CT, experienced worse baseline QOL, and had worse 12-month survival. Intensive medical nutrition therapy was not associated with differences in CT associated toxicity. Novel nutritional interventions are still needed to improve pt outcomes.

Lung cancer is a major global health problem, as it is the leading cause of cancer-related deaths worldwide. Major advances in the identification of key mutational alterations have led to the development of molecularly targeted therapies, whose efficacy has been limited by emergence of resistance mechanisms. US Food and Drug Administration (FDA)-approved therapies targeting angiogenesis and more recently immune checkpoints have reinvigorated enthusiasm in elucidating the prognostic and pathophysiological roles of the tumour microenvironment in lung cancer. In this Review, we highlight recent advances and emerging concepts for how the tumour-reprogrammed lung microenvironment promotes both primary lung tumours and lung metastasis from extrapulmonary neoplasms by contributing to inflammation, angiogenesis, immune modulation and response to therapies. We also discuss the potential of understanding tumour microenvironmental processes to identify biomarkers of clinical utility and to develop novel targeted therapies against lung cancer.

Although didymin, a dietary flavonoid glycoside from citrus fruits, known to be a potent antioxidant with anti-cancer activities, its role in angiogenesis is not known. In this study, we examined the effect of didymin on VEGF-induced angiogenesis in vitro and in vivo models. Our results suggest that treatment of human umbilical vein endothelial cell (HUVECs) with didymin significantly prevented the VEGF-induced cell proliferation, migration, and invasion. Further, didymin significantly prevented the VEGF-induced endothelial tube formation in culture. Didymin also attenuated the VEGF-induced generation of ROS, activation of NF-κB and the expression of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin in HUVECs. Further, didymin also prevented the VEGF-induced microvessel sprouting in ex vivo mouse aortic rings. Most importantly, didymin significantly prevented the invasion of endothelial cells and formation of blood capillary-like structures in Matrigel plug model of angiogenesis in mice. Thus, our results suggest a novel antiangiogenic efficacy of didymin in addition to its reported anti-cancer properties, which warrant further development of this agent for cancer therapy.

The recent emergence of targeted and immunotherapeutic agents has dramatically changed the management for patients with non-small cell lung cancer (NSCLC). Despite these advances, lung cancer is not exempt from the challenges facing oncology drug development, including the huge financial cost and the time required for drug implementation. Repositioning noncancer therapies with potential antineoplastic properties into new therapeutic niches is an alternative treatment strategy offering the possibility of saving money and time and improving outcomes. The goal of such a strategy is to deliver an effective drug with a favorable toxicity profile at a reduced cost. Preclinical models and observational data have demonstrated promising activity for many of these agents, and they are now being studied in prospective trials. We review the relevant published data regarding the therapeutic effects of metformin, statins, nonsteroidal anti-inflammatory drugs, β-blockers, and itraconazole in NSCLC, with a focus on the putative mechanisms of action and clinical data. As these drugs are increasingly being tested in clinical trials, we aim to highlight the salient challenges and future strategies to optimize this approach. Implications for Practice: The staggering failure rates, exorbitant costs, and lengthy approval process associated with drug development in lung cancer warrants exploration of alternative strategies. The repositioning of approved noncancer medications to treat non-small cell lung cancer (NSCLC) represents a unique opportunity to improve outcomes by delivering an effective drug at lower costs with manageable toxicity. Several such agents have demonstrated antineoplastic activity and are being studied in NSCLC patient populations. The present review highlights the relevant literature regarding these agents’ therapeutic effects and reports on the challenges in implementing this strategy moving forward, including a discussion of ongoing phase I, II, and III trials.

Although we have shown earlier that aldose reductase (AR) inhibitors prevent colorectal cancer cell (CRC) growth in culture as well as in nude mice xenografts, the mechanism(s) is not well understood. In this study, we have investigated how AR inhibition prevents CRC growth by regulating the mitochondrial biogenesis via Nrf2/HO-1 pathway. Incubation of CRC cells such as SW-480, HT29, and HCT116 with AR inhibitor, fidarestat that non-covalently binds to the enzyme, increases the expression of Nrf2. Further, fidarestat augmented the EGF-induced expression of Nrf-2 in CRC cells. Fidarestat also increased the Nrf2 -DNA binding activity as well as expression of HO-1 and NQO1 and activation of SOD and catalase in SW480 cells. Similarly, in nude mice xenograft tumor tissues, Nrf2 and HO-1 levels were significantly higher in fidarestat-treated mice compared to controls. Further, stimulation of CRC cells with EGF in the presence of fidarestat increased the mRNA levels of PGC-1α, Nrf1 and TFAM and protein levels of PGC-1α, TFAM and COX-IV and decreased the mitochondrial DNA damage as measured by 8-hydroxy-2′-deoxyguanosine levels. AR inhibitor also modulated the phosphorylations of AMPK and mTOR and expression of p53 in EGF-treated cells. Collectively, our results indicate that AR inhibitor prevents CRC growth by increasing mitochondrial biogenesis via increasing the expression of Nrf2/HO-1/AMPK/p53 and decreasing the mitochondrial DNA damage.

Type-2 diabetes and obesity-related metabolic abnormalities are major risk factors for the development of colon cancer. In the present study, we examined the effects of polyol pathway enzyme aldose reductase (AR) inhibitor, fidarestat, on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db obese mice. Our results indicate that fidarestat given in the drinking water caused a significant reduction in the total number of colonic premalignant lesions in the AOM treated obese mice. Further, the expression levels of PKC-β2, AKT, COX-2 and iNOS in the colonic mucosa of AOM-treated mice were significantly decreased by fidarestat. The serum levels of IL-1α, IP-10, MIG, TNF-α and VEGF are significantly suppressed in AOM + fidarestat treated obese mice. Fidarestat also decreased the expression of COX-2, iNOS, XIAP, survivin, β-catenin and NF-κB in high glucose-treated HT29 colon cancer cells. In conclusion, our results indicate that fidarestat inhibits the development of colonic premalignant lesions in an obesity-related colon cancer and is chemopreventive to colorectal carcinogenesis in obese individuals.

The development of intratumoral hypoxia, a hallmark of rapidly progressing solid tumors, renders tumor cells resistant to chemotherapy and radiation therapy. We have recently shown that inhibition of aldose reductase (AR), an enzyme that catalyzes the reduction of lipid aldehydes and their glutathione conjugates, prevents human colon cancer cell growth in culture as well as in nude mouse xenografts by inhibiting the NF-κB-dependent activation of oxidative stress-mediated inflammatory and carcinogenic markers. However, the role of AR in mediating hypoxic stress signals is not known. We therefore investigated the molecular mechanisms by which AR inhibition prevents the hypoxia-induced human colon cancer cells growth and invasion. Our results indicate that AR inhibition by the pharmacological inhibitor fidarestat or ablation by AR-specific siRNA prevents hypoxia-induced proliferation of HT29, SW480, and Caco-2 colon cancer cells. Furthermore, hypoxia-induced increase in the level of HIF-1α in colon cancer cells was significantly decreased by AR inhibition. During hypoxic conditions, treatment of HT29 cells with the AR inhibitor fidarestat significantly decreased the expression of vascular endothelial growth factor, a down target of HIF-1α, at both mRNA and protein levels and also prevented the activation of PI3K/AKT, GSK3β, Snail, and lysyl oxidase. Furthermore, inhibition of hypoxia-induced HIF-1α protein accumulation by AR inhibition was abolished in the presence of MG132, a potent inhibitor of the 26 S proteasome. In addition, AR inhibition also prevented the hypoxia-induced inflammatory molecules such as Cox-2 and PGE2 and expression of extracellular matrix proteins such as MMP2, vimentin, uPAR, and lysyl oxidase 2. In conclusion, our results indicate that AR mediates hypoxic signals, leading to tumor progression and invasion.

143 Background: There is an emerging evidence that the gut microbiome may influence anti-tumor immune response during treatment with checkpoint inhibitors (CPIs). A number of factors can cause gut microbiome dysbiosis including the use of antibiotics (ABX). We hypothesized that the use of ABX during immunotherapy can adversely affect the efficacy of CPIs. We explored the prevalence of ABX use amongst patients (pts) using CPIs, and whether the use of ABX influences the response to CPIs. Methods: We performed an institutional retrospective review of all the pts treated with CPIs from 2/2015-3/2018. A patient was considered to have used ABX if he or she was prescribed ABX within 6 months before or after, initiating CPIs. Statistical analysis was done using logistic regression with overall response rate (ORR) (CR, PR and SD) as the outcome. 9 separate analyses were done: one for each temporality (30 days, 60 days, 6 months) and use-order (before, after, neither) combination. Odds ratios for ORR as univariate analysis, and adjusted for age and sex; and adjusted for age, sex, and tumor type were calculated. Results: Out of 242 pts, 111 were lung, 36 bladder, 35 renal, 16 gastrointestinal and 44 other cancers. 50% (121) pts received nivolumab, 28% (68) pembrolizumab and 21% (52) atezolizumab. 75%, 46% and 32% of the pts received ABX within 6 months, 60 days and 30 days of starting CPIs. Only ABX use in the first 30- or 60-days following CPI initiation was associated with inferior ORR (OR 0.42 [95% CI:0.23,0.76] p = 0.005 for 60-days). ABX use prior to initiation of CPI at any time point, or ABX use in the first 6 months of CPI use did not impact CPI efficacy, Table 1. Conclusions: ABX use within the first 60 days of starting CPI therapy is prevalent (32%). This study suggests that the use of ABX within 60 days following initiation of CPIs significantly negatively impacts the ORR. Unnecessary usage of ABX should be avoided, especially during the early phase of starting CPIs. [Table: see text]

Introduction: Hypermethylation of key tumor suppressor genes plays an important role in lung carcinogenesis. The purpose of this study is to explore the therapeutic potential of regional administration (via the airways) of the demethylating agent 5-azacytidine (5-Aza) for the treatment of early lung cancer. Patients and Methods We administered 5-Aza solution directly into the trachea in imprinting control region (ICR) mice (to study its toxicity) and in nude mice bearing orthotopic human lung cancer xenografts (to assess its antitumor activity). Results In vitro, 5-Aza inhibited the growth of human lung cancer cell lines H226, H358, and H460 in a dose-dependent manner. The concentrations to inhibit cell growth by 50% (IC 50 ) were about 0.6-4.9 μg/mL. 5-Azacytidine reversed hypermethylation in the promoter of tumor suppressor gene RASSF1a in the H226 cells at a 6000-fold lower concentration than its IC 50 . In animal studies, intratracheal (I.T.) administration of 90 mg/kg 5-Aza (the maximum tolerated dose of 5-Aza intravenous injection [I.V.]) resulted in moderate pulmonary toxicity and 5-fold reduced myelosuppression compared with the same dose of I.V. 5-Aza. Using an optimized multiple dose schedule, I.T. 5-Aza was about 3-fold more effective than I.V. 5-Aza in prolonging the survival of mice bearing orthotopic H460 and H358 xenografts, and did not cause any detectable toxicity. Conclusion 5-Azacytidine can reverse the hypermethylation in the human lung cancer cell lines at a nontoxic dose. Regional administration to the airways enhances the therapeutic index of 5-Aza by 75-fold. The potential of regional administration of 5-Aza (including by aerosolization) for the treatment of advanced bronchial premalignancy deserves further investigation.

Immune checkpoint inhibitors such as anti-programmed death 1 (PD1) and anti-PD-L1 monoclonal antibodies though have great potential for cancer immunotherapy, the acquired drug resistance and unwanted immune side effects limit their use. However, the combination therapy using conventional drugs and immune checkpoint inhibitors not only synergistically increase anti-tumor responses but it also reduces immune-associated adverse events. In our earlier studies, we have shown that inhibition of polyol pathway enzyme, aldose reductase (AR) prevents colon cancer growth and metastasis in culture as well as in mouse models. In this study, we investigated whether fidarestat, a potent AR inhibitor that has already gone through Phase-III clinical trial for diabetic neuropathy and found to safe for human use, can synergize with anti PD-1 to increase antitumor activity and decrease immune-associated adverse effects. Mice bearing syngeneic CT26 colon carcinoma tumors were treated with anti PD-1 alone or in combination with fidarestat, and the tumor growth was monitored. Our results indicate that fidarestat in combination with anti-PD1 synergistically increases tumor regression within 28 days of treatment. Further, a combination of fidarestat and anti-PD1 significantly increased the CD8+ T cells and natural killer (NK) cells and decreased the Myeloid-derived suppressor cells (MDSCs) at tumor milieu. Fidarestat plus anti-PD1 also increased CD8+ T cell and NK cell effector functions as determined by IFNg and granzyme B. Systemically, combination therapy also increased the number of CD8+ T cells and NK cells and decreased MDSCs in the mouse spleens. Further, the effector functions of CD8+ T cells and NK cells were increased with the combination treatment in mice spleens. Moreover, the levels of various inflammatory cytokines and chemokines in the serum and liver were significantly less in fidarestat plus anti-PD1 -treated mice as compared to anti-PD1 alone -treated mice indicating that fidarestat rescues immune-mediated adverse events in various organs. Thus, our results suggest that by regulating the number as well as effector function of CD8+ T cells, NK cells and MDSCs, AR inhibitor in combination with anti-PD1 would not only regress the tumor growth but would also provide a long-lasting specific immunity against colon cancer, which would decrease the chances of tumor recurrence. Citation Format: Ashish Saxena, Himangshu Sonowal, Satish K. Srivastava, Kota V. Ramana. Combined treatment of aldose reductase inhibitor, fidarestat, and anti-PD1 antibodies synergistically prevents T cell- and NK cell-mediated colon cancer growth in murine models [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 1682.

Anthracycline class of chemotherapeutic drugs such as doxorubicin and daunorubicin has been known to be effective in the therapy of various types of cancer. However, in certain cancers such as colon cancer, the use of anthracyclines is restricted due to acquired drug resistance, which requires high amounts of drugs that cause severe toxic side effects. We have recently demonstrated that aldose reductase (AR) inhibitor, fidarestat, increases the sensitivity of doxorubicin towards colon cancer cells death in vitro and in vivo and decreases cardiotoxicity. However, the mechanisms through which AR inhibitor prevents doxorubicin-induced toxicity is not understood. We have examined how AR inhibition prevents doxorubicin-induced endothelial and cardiac toxicity along with inflammatory response in in vitro and in vivo models. Our results demonstrate that fidarestat prevents doxorubicin-induced oxidative stress, endothelial cell death, and monocyte adhesion. Further, fidarestat prevents the activation of NF-κB-dependent expression of endothelial cell markers ICAM-1 and VCAM-1 and expression of various inflammatory cytokines and chemokines in human vascular endothelial cells. In addition, fidarestat prevented the doxorubicin-induced decrease in eNOS and increase in iNOS expression. Similarly, AR inhibition prevented doxorubicin-induced endothelial dysfunction, cardiac hypertrophy, and cardiomyocyte injury in mice. Fidarestat also prevented doxorubicin-induced activation of monocytes and macrophages to proinflammatory phenotype in culture as well as in mouse models. Thus, our results suggest that by preventing doxorubicin-induced inflammatory as well as oxidative stress signals, fidarestat decreases cardiotoxic effects of doxorubicin. This is a novel adjuvant therapy of cancer using aldose reductase inhibitors with anthracyclines that would increase cancer therapeutic efficacy and decrease cardiotoxic side effects. Citation Format: Himangshu Sonowal, Ashish Saxena, Kota V. Ramana, Satish K. Srivastava. Aldose reductase inhibition prevents doxorubicin-induced inflammatory response in endothelial cells and macrophages [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 5815.

Abnormal proliferation of vascular smooth muscle cells (VSMC) is a key feature of development of cardiovascular complications, atherosclerosis, and restenosis. Patients with diabetes have higher risk for restenosis after coronary angioplasty than nondiabetic patients due to hyperglycemia-induced release of cytokines such as TNF-alpha. However, the molecular mechanisms regulating VSMC proliferation remain unclear. Herein, we report that inhibition of the polyol pathway enzyme aldose reductase (AR) prevents high glucose (HG)- and/or TNF-alpha-induced VSMC proliferation by accumulating cells at the G1 phase of the cell cycle. Treatment of VSMC with AR inhibitor sorbinil prevented HG- as well as TNF-alpha-induced phosphorylation of retinoblastoma protein and activation of E2F-1. Inhibition of AR also prevented HG- and TNF-alpha-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G1/S transition regulatory proteins such as cyclin D1, cyclin E, cdk-4, c-myc, and proliferative cell nuclear antigen. More importantly, inhibition of AR prevented the increased expression of E2F-1 and proliferative cell nuclear antigen in diabetic rat aorta. Treatment of VSMC with the most abundant and toxic lipid aldehyde 4-hydroxy-trans-2-nonenal (HNE) or its glutathione conjugate [glutathionyl (GS)-HNE] or AR-catalyzed product of GS-HNE, GS-1,4-dihydroxynonane, resulted in increased E2F-1 expression. Inhibition of AR prevented HNE- or GS-HNE-induced but not GS-1,4-dihydroxynonane-induced up-regulation of E2F-1. Collectively, these results show that AR could regulate HG- and TNF-alpha-induced VSMC proliferation by altering the activation of G1/S-phase proteins such as E2F-1, cdks, and cyclins. Thus, inhibition of AR may be a useful therapeutic approach in preventing vascular complications.

The role of single-agent nab-paclitaxel in relapsed or platinum-refractory advanced non-small cell lung cancer (NSCLC) has not been well reported in Western populations. We reviewed our own institution's experience using nab-paclitaxel in these settings. We analyzed the records of stage IV NSCLC patients with relapsed or platinum-refractory disease treated with single-agent nab-paclitaxel at Weill Cornell Medical College between October 2008 and December 2013. The primary endpoint of the study was treatment failure-free survival (TFFS), defined as the time from the start of nab-paclitaxel therapy to discontinuation of the drug for any reason. The best overall response was recorded for each patient, and overall response and disease control rates were calculated. Thirty-one stage IV NSCLC patients received a median of 4 cycles (range 1-40) of nab-paclitaxel. Dose reduction or drug discontinuation due to toxicity occurred in 10 patients, mainly because of grade 2/3 fatigue or peripheral neuropathy. The overall response rate was 16.1 %, and the disease control rate was 64.5 %. Median TFFS was 3.5 months (95 % CI 1.3-5.3 months). No statistically significant difference in TFFS based on line of therapy or prior taxane exposure was identified. There was a statistically significant decrease in TFFS for patients with non-adenocarcinoma histology, although there were only five patients in this group. There was a trend toward reduction in the risk of treatment failure with increasing age. One patient remained on nab-paclitaxel therapy for over 3 years. Single-agent nab-paclitaxel was well tolerated and demonstrated efficacy in advanced NSCLC patients with relapsed or platinum-refractory disease. Further prospective clinical trials with nab-paclitaxel in these settings are warranted.

CTCs are used as a surrogate source of tumor material in solid tumors. Clinical applications of CTCs as liquid biopsy comprise the monitoring of molecular alterations during tumor progression and dynamic evaluation of molecular markers of treatment response. The FDA-cleared method to isolate CTCs in cancer patients (Cell Search) is based on positive selection of EpCAM expressing cells. However, this approach performs poorly in non-small cell lung cancer (NSCLC) as it identifies CTCs in only 7% of the subjects, failing to show any prognostic relevance. Down-regulation/loss of epithelial markers to isolate (EpCAM) and identify (cytokeratin, CK) CTCs could in part explain the low CTC yield obtained in NSCLC with approaches based on epithelial markers expression. To overcome this challenge we used size-based CTC enrichment (ISET filters) from NSCLC patients’ peripheral blood. As a positive identifier of CTCs we used transferrin receptor 1 (TfR) which is a cell membrane-associated protein, that mediates intracellular iron uptake, and which is expressed at low levels in many normal tissues but over-expressed in cancer cells. We first analyzed TfR protein expression by immunofluorescence in a panel of NSCLC cell lines and in healthy donor leukocytes. While all NSCLC cells lines analyzed were positive for TfR expression, none of the leukocyte expressed the receptor. Moreover, TfR expression was detected also in EpCAM negative NSCLC cell lines. To determine the clinical applicability of this novel CTC identifier, we determined TfR expression in CTCs isolated from peripheral blood of 35 metastatic NSCLC patients using the ISET filter technology. The isolated CTCs were stained for TfR, CK, CD45 and DAPI. For each patient, one additional ISET filter was stained with Giemsa for morphologic analysis by a pathologist. By using the classic panel of CTC identifiers markers (CK+/CD45-/DAPI+), CTCs were identified in 4/34 (11%) patients, while by using TfR as positive identifier (TfR+/CD45-/DAPI+) CTCs were identified in 31/35 (88%) subjects. The morphologic review of Giemsa stained filters confirmed the presence of tumor cells in 28/34 (82%) samples [0-217 CTCs/sample]. Interestingly, patients with > 6 TfR+ CTCs had a worse overall survival (OS) than patients with < 6 TfR+ CTCs [p=0.048 Log Rank (Mantel-Cox)]. OS did not significantly differ using the same cutoff with CTCs defined based on CK or Giemsa staining. Overall, our data indicate that TfR is a promising biomarker for the detection of CTCs in NSCLC CTCs, superior to CK or EpCAM. Our data also suggest that TfR may potentially identify CTCs subpopulations with a significant prognostic role in NSCLC. We are currently isolating TfR+ CTCs from early stage and metastatic NSCLC patients for further molecular characterization and determination of clinical significance. Citation Format: Giuseppe Galletti, Galatea Kallergi, Ashish Saxena, Despoina Aggouraki, Christos Stournaras, Vassilis Georgoulias, Timothy E. McGraw, Nasser Altorki, Paraskevi Giannakakou. Transferrin receptor 1 (TfR) as marker for circulating tumor cells (CTCs) identification in NSCLC [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 1713. doi:10.1158/1538-7445.AM2017-1713

Colorectal cancer (CRC) is the third most common form of cancer and the second leading cause for cancer deaths. Several epidemiological, in vitro and in vivo studies suggest that Estrogen receptor β (ERβ) could be a possible target for CRC prevention and treatment, but the potential mechanism is not demonstrated. ERβ is expressed in intestinal epithelial cells, as well as in some intestinal immune cells and other tissues. Previous studies have seen increased CRC after full ERβ knockout, but it is not clear if this is mediated by intestinal ERβ. The specific aim of our study is to determine the role of intestinal ERβ during colon cancer development in vivo. We deleted ERβ specifically in the intestine epithelia of mice (iERβKO), and induced CRC using Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) treatment. After 9 weeks treatment, all mice presented colitis and two iERβKO mice but none of the wild-type (WT) mice had developed tumors. qPCR showed that the expression of pro-inflammatory markers was higher in iERβKO treated mice than in untreated or WT mice, Immunohistochemistry (IHC) of proliferative markers showed that iERβKO exhibited higher cell proliferation in the top of the crypts compared to controls. After 16 weeks treatment, all mice developed tumors. qPCR showed that the expression of mesenchymal markers was higher in treated iERβKO mice compared to treated WT mice. Our results demonstrate that intestinal ERβ attenuates colon cancer-related pathways. Citation Format: Linnea Pettersson, Ashish Saxena, Trang Vu, Jan-Åke Gustafsson, Amena Archer, Cecilia Williams. Intestinal estrogen receptor beta attenuates colon cancer signaling pathways in vivo [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 2819. doi:10.1158/1538-7445.AM2017-2819

Background: Chronic inflammation is a risk factor of colorectal cancer (CRC), and the microbiota of the gut is important in both colitis and carcinogenesis. Estrogen receptor beta (ERβ) is the predominant nuclear receptor in the colon, and is downregulated in CRC. Here, we explore the role of colitis and ERβ signaling with respect to the microbiota diversity. Methods: Intestinal ERβ-/- mice were generated using the Cre-loxP system. WT and knockout mice were treated with AOM/DSS inducing colitis (at 9 weeks) and CRC (at 16 weeks). After 9 and 16 weeks, stool pellets were collected from treated and untreated mice (n=39), and DNA was extracted. Library preparation and 16S rRNA-sequencing was performed using the illumina Miseq platform with specific primers targeting the V3-V4 regions, and QIIME software was used to analyze microbiota diversity. Sequencing results were validated by qPCR Results: High-throughput 16S rRNA gene sequencing demonstrated that treatment induced significant differentiation of both alpha (p=0.01) and beta (p=0.01) microbiota diversity. Alpha diversity indicators implied a significant reduction of microbiota richness and evenness after treatment. qPCR confirmed that 9-weeks treatment increased OTUs affiliated with Bifidobacteria (p=0.03), Bacteroidetes (p=0.04), Lactobacilli (p=0.0014), Cyanobacteria (p=0.0023) and Gammaproteobacteria (p=0.05) in WT mice. After 16 weeks treatment, 16s rRNA sequencing show enrichment of Tenercutes, Proteobacteria and Cyanobacteria in all tumor-bearing mice (treated WT and ERβ-/-), while Verrucomicrobia increased in untreated mice (No tumors). Further treated ERβ-/- but not WT mice displayed significant reduction of Gammaproteobaceria (p=0.0083). Conclusion: Our data demonstrate a clear effect of colitis-induced treatment on the microbiota, and that ERβ attenuates this response. Lactobacilli and Bifidobacteria has, for example, been shown to attenuate inflammatory response. We note an increase in Lactobacilli and Bifidobacteia after treatment in WT mice, but not in ERβ-/- mice. This can result in increased colitis in ERβ-/- mice and help explain the increased expression of pro-inflammatory cytokines we note in treated ERβ-/- mice. Citation Format: Ahmed F. Ibrahim, Quentin Thomas, Ashish Saxena, Cecilia Williams. Inflammation and estrogen receptor beta modulate bacterial diversity of the gut [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 4920. doi:10.1158/1538-7445.AM2017-4920

Inhibition of polyol pathway enzyme aldose reductase (AR) has been shown to prevent colon cancer cells growth in culture and in nude mice xenografts. However, the role of AR in the mediation of growth factor-induced colon cancer cells growth is not well understood. In this study, we have investigated how AR inhibition prevents tumour growth via regulation of microRNA (miR)-21-mediated programmed cell death 4 (PDCD4) expression in colon cancer cells in in vitro and in vivo. Treatment of colon cancer cells (HT29, SW480 and Caco-2) with epidermal growth factor (EGF) caused increased expression of miR-21 and inhibition of AR prevented it. Further, AR inhibition also increased PDCD4, a putative target of miR-21 in human colon cancer cells. Inhibition of AR also prevented EGF-induced phosphorylation of PDCD4. Treatment of HT29 cells with AR inhibitor, fidarestat, prevented the EGF-induced phosphorylation of mammalian target of rapamycin (mTOR), regulatory associated protein of mTOR (Raptor), eukaryotic initiation factor 4E (eIF4E), p70 S6 kinase (S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and increased the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Similarly, in nude mice xenograft tissues, PDCD4 and 4E-BP1 levels were significantly higher in AR inhibitor-treated mice compared to controls. Collectively, these results indicate that AR inhibition prevents growth factor-induced colon cancer growth by down-regulating miR-21 expression and increasing PDCD4 levels through the reactive oxygen species (ROS)/AMPK/mTOR/AP1/4E-BP1 pathway.

Aims: We have shown earlier that inhibition of aldose reductase (AR), an oxidative stress-response protein, prevents colon cancer cell growth in vitro and in vivo. Changes in microribonucleic acid (miR) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanisms by which AR regulates miR expression and their dependent mitogenic effects in cancer cells are not known. Therefore, we investigated how AR regulates growth factor-induced expression of miRs and growth of colon cancer cells. Results: Inhibition of AR significantly downregulated growth factor-induced miR-21 expression in human colon cancer cells, HT29, SW480, and Caco-2. Further, AR inhibition also increased phosphatase and tensin homolog (PTEN) (a direct target of miR-21) and forkhead box O3A (FOXO3a) in colon cancer cells. Our results obtained with HT29 cells ablated with FOXO3a siRNA showed increased activator protein-1 (AP-1) activation and miR-21 expression, indicating that FOXO3a represses miR-21 via AP-1 inactivation. Inhibition of AR also prevented the epidermal growth factor-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), c-Jun, c-Fos, PTEN, and FOXO3a, and deoxyribonucleic acid (DNA)-binding activity of AP-1. More importantly, in human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. Innovation: These findings demonstrate a novel role of AR in the regulation of miR-21 and its target PTEN in growth factor-induced colon cancer cell growth. Conclusions: Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer growth by modulating miR-21, PTEN, and FOXO3a expression through reactive oxygen species (ROS)/PI3K/AKT/AP-1. Antioxid. Redox Signal. 18, 1249–1262.

e18029 Background: NabP is a formulation of P designed to reduce toxicity of therapy and increase drug delivery to tumor cells. An established therapy for st4 breast cancer, single-agent nabP has also been shown to produce responses in frontline therapy of st4 NSCLC. Its role in relapsed NSCLC has not been well reported. Methods: We analyzed the records of st4 NSCLC patients (pts) treated with single-agent nabP at our institution. Cases were assessed with regard to age, sex, KPS, histology, line of systemic therapy, prior taxane exposure, # of cycles administered, dose attenuation, and progression-free survival (PFS) estimated by Kaplan-Meier analysis. Standard dosing of nabP was considered to be 260 mg/m2 IV Q 21 days. Disease was considered to have progressed on the date of any clinical assessment (typically CT scan) that led the treating oncologist to conclude that disease had progressed. All pts completed nabP therapy except 1, who was assessed at 427 days of follow up. Results: From Oct 2008 to Jan 2012, 15 st4 NSCLC pts (median age 67, range 48 to 82; 8 F, 7 M; KPS 60-80%; 12 adenocarcinoma, 2 squamous cell, 1 unspecified) received a median of 3 cycles (range 1-17) of single-agent nabP. All pts received at least 1 prior line of systemic therapy in the st4 setting: 6 received nabP as 2nd line therapy, 4 as 3rd line, and 5 as 4th line. 5 pts had also received prior chemotherapy for early stage NSCLC. 9 pts were taxane-naïve; 6 had received prior taxane therapy. 10 pts began nabP at standard dose; 5 began on an attenuated dose or schedule. Subsequent dose reduction occurred in 3 pts due to toxicity (fatigue, myelosuppression). Only 1 patient discontinued treatment due to toxicity (myalgias, arthralgias). Median PFS for all pts was 82 days (95% CI 35 to 205 days). Older age reduced risk of progression [hazard ratio 0.93 per 1-year age increase (95% CI 0.86 to 0.99), P=0.04]. Prior taxane exposure decreased PFS (median 39 vs. 113 days, P=0.07 by log-rank test). PFS did not vary by KPS, histology, or line of therapy. Conclusions: Single-agent nabP administered in relapsed St4 NSCLC is well tolerated and associated with extension of PFS, particularly in older and taxane-naïve pts.

We have shown earlier that the inhibition of aldose reductase (AR), a NADPH-dependent aldo-keto reductase, prevents growth factor-induced proliferation of colon cancer cells in vitro and in vivo. Recent studies indicate that changes in microRNA (miRNA) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanism(s) by which AR regulates miRNA expression and their dependent mitogenic effects in cancer cells is not known. Therefore, we investigated how AR regulates growth factor -induced expression of miRNAs in colon cancer cells. Our results indicate that inhibition of AR significantly down-regulated growth factor-induced miR-21 expression in HT29, SW480 and Caco-2 human colon cancer cells. Further, AR inhibition also increased PTEN, a direct target of miR-21 that contributes cancer cell growth and invasion. Inhibition of AR also prevented the EGF-induced phosphorylation of PTEN and FOXO3a, which prevents cell proliferation. Treatment of human colon cancer cells with AR inhibitor, fidarestat, prevented the EGF-induced DNA binding activity of AP1, phosphorylation of PI3K, AKT, c-Jun and c-Fos. More importantly, in HT29 human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer cell proliferation by increasing miR-21 expression and inactivating or decreasing PTEN and FOXO3a through the ROS/PI3K/AKT/AP1 pathway, indicating that AR inhibitors could be used for the prevention/therapy of colon carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2296. doi:1538-7445.AM2012-2296

Chronic inflammatory diseases and oxidative stress are major risk factors of colorectal cancer (CRC), the third most common cause of death among cancer patients. Although several agents such as antioxidants, plant products and nutritional supplements have been shown to have some chemopreventive effects on colon cancer, none are very effective to completely. Recent studies indicate that a fat soluble analogue of vitamin B1, benfotiamine is an excellent antioxidant and anti-inflammatory. However, the chemo-preventive or -therapeutic efficacy of benfotiamine in preventing CRC is not known. Our hypothesis is that benfotiamine through its anti-oxidant and anti-inflammatory properties could be very effective in preventing CRC. We therefore investigated the role of benfotiamine in the prevention of carcinogenic signals leading to CRC in cell culture as well as murine models of CRC. Our results show that treatment of human colon cancer cells (HT-29 and Caco-2) in culture with benfotiamine prevented the cancer cell proliferation. Further, benfotiamine also prevented the growth of human adenocarcinoma cells (SW480) -induced tumor growth in nude mice xenografts. Our studies also indicate that benfotiamine prevented NF-kB-dependent survival signals and activated caspase-3 dependent apoptotic signals in colon cancer cells. Further, benfotiamine supplementation also suppressed azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation in mice and AOM-induced inflammatory changes such as activation of Cox-2 and iNOS, and carcinogenic changes such as expression of cyclin D1 and β-catenin in mice colons. In conclusion, our results indicate that benfotiamine prevents colon cancer cell growth in culture and nude mice xenografts as well as formation of ACF in AOM-treated mice. Thus benfotiamine supplementation could be used as an excellent chemopreventive agent for the treatment of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2397. doi:10.1158/1538-7445.AM2011-2397

We have recently shown that Aldose Reductase (AR) known to be involved in oxidative stress-signaling, initiated by inflammatory markers, prevents human colon cancer cell growth in culture as well as in nude mice xenografts. Inhibition of AR also prevents azoxymethane-induced aberrant crypt foci formation in mice. In order to understand the chemopreventive mechanism of AR inhibition in colon cancer, we have investigated the role of AR in the mediation of angiogenic signals in vitro and in vivo models. Using human umbilical vein endothelial cells (HUVEC), we have investigated the effect of AR inhibition on VEGF- and bFGF- induced tube as well as spheroid formation in in vitro angiogenesis. We have also determined the invasion and migration of HUVEC by inhibition of AR. Further, to determine the in vivo efficacy of AR inhibition on angiogenesis, matrigel plug assay was performed using a rat model. Our results show that inhibition of AR significantly prevented the VEGF- and bFGF -induced proliferation and generation of reactive oxygen species in HUVEC. Further, AR inhibition also prevented the VEGF- and bFGF- induced secretion/expression of IL-6, MMP1, MMP9, ICAM, and VCAM. Matrigel plug model of angiogenesis in rats showed that inhibition of AR prevented the infiltration of blood cells, invasion, migration and formation of capillary like structures, and expression of blood vessels markers CD31 and vWF. Thus, our results demonstrate that AR inhibitors could be novel drugs for cancer chemoprevention by inhibiting angiogenesis. 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 1384.