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Gastrointestinal (GI) cancers are one of the leading causes of death worldwide. Although various approaches are implemented to improve the health condition of GI patients, none of the treatment protocols promise for eradicating cancer. However, a treatment mechanism against any kind of disease condition is already existing executing inside the human body. The 'immune system' is highly efficient to detect and destroy the unfavourable events of the body including tumor cells. The immune system can restrict the growth and proliferation of cancer. Cancer cells behave much smarter and adopt new mechanisms for hiding from the immune cells. Thus, cancer immunotherapy might play a decisive role to train the immune system against cancer. In this review, we have discussed the immunotherapy permitted for the treatment of GI cancers. We have discussed various methods and mechanisms, periodic development of cancer immunotherapies, approved biologicals, completed and ongoing clinical trials, role of various biopharmaceuticals, and epigenetic factors involved in GI cancer immunotherapies (graphical abstract Figure 1).

Pancreatic cancer (PanCa) is a highly lethal disease with a poor 5 year survival rate, less than 7%. It has a dismal prognosis, and more than 50% of cases are detected at an advanced and metastatic stage. Gemcitabine (GEM) is a gold standard chemotherapy used for PanCa treatment. However, GEM-acquired resistance in cancer cells is considered as a major setback for its continued clinical implementation. This phenomenon is evidently linked to de novo lipid synthesis. PanCa cells rely on de novo lipid synthesis, which is a prime event in survival and one of the key drivers for tumorigenesis, cancer progression, and drug resistance. Thus, the depletion of lipogenesis or lipid metabolism can not only improve treatment outcomes but also overcome chemoresistance, which is an unmet clinical need. Toward this effort, our study reports a unique paclitaxel–poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PPNPs) formulation which can target lipid metabolism and improve anticancer efficacy of GEM in PanCa cells. PPNPs inhibit excessive lipid formation and alter membrane stability with compromised membrane integrity, which was confirmed by Fourier transform infrared and zeta potential measurements. The effective interference of PPNPs in lipid metabolic signaling was determined by reduction in the expression of FASN, ACC, lipin, and Cox-2 proteins. This molecular action profoundly enhances efficacy of GEM as evident through enhanced inhibitory effects on the tumorigenic and metastasis assays in PanCa cells. These data clearly suggest that the ablation of lipid metabolism might offer an innovative approach for the improved therapeutic outcome in PanCa patients.

Utilization of safe cytotoxic agents with precise anticancer activity is considered as the prime focus of cancer therapeutics research. A greater incentive for such agents arises from the molecules/drugs that are already being used for other indications. Ormeloxifene (ORM) is a nonsteroidal, nonhormonal selective estrogen receptor modulator (SERM), which has been in human use for contraception purposes. Although in the recent past, many reports have suggested its emerging role as an anticancer agent, no significant attention was paid toward generating simple and safe nanoformulation(s) for improved therapeutic activity and tumor cell-specific delivery. Our aim is to develop nanoformulation(s) of ormeloxifene to improve its targeted delivery in tumor cells. We developed ormeloxifene nanoformulation(s) by utilizing various biocompatible polymers. The optimized formulations with pluronic polymers F127 and F68 show improved nanoparticle characteristics. These formulations show enhanced cellular uptake that allows ormeloxifene's intracellular availability. We further evaluated its improved anticancer activity by performing cell proliferation, flow cytometry, and immunoblotting assays. Overall, this study confirms possible novel nanoformulation(s) of ormeloxifene to be evolved as a new therapeutic modality for cancer treatment.

Stress management is becoming very important part of cancer patient care. Chronic stressors lead to boost tumorigenesis and promote cancer development, recurrence, and drug resistant leading to poor health outcomes. The Hypothalamic-Pituitary-Adrenal (HPA) axis, which is activated by stress, also regulates Hypothalamic-Pituitary-Thyroid (HPT) axis. Stress related changes in immune function and inflammatory response also leads to reduced immune surveillance resulting in tumorigenesis. This article explores the hormonal axis impacted by stress and how chronic stress can lead to poor outcome of a cancer patient.

Cellular senescence is one of the prevailing issues in cancer therapeutics that promotes cancer relapse, chemoresistance, and recurrence. Patients undergoing persistent chemotherapy often develop drug-induced senescence. Docetaxel, an FDA-approved treatment for prostate cancer, is known to induce cellular senescence which often limits the overall survival of patients. Strategic therapies that counter the cellular and drug-induced senescence are an unmet clinical need. Towards this an effort was made to develop a novel therapeutic strategy that targets and removes senescent cells from the tumors, we developed a nanoformulation of tannic acid−docetaxel self-assemblies (DSAs). The construction of DSAs was confirmed through particle size measurements, spectroscopy, thermal, and biocompatibility studies. This formulation exhibited enhanced in vitro therapeutic activity in various biological functional assays with respect to native docetaxel treatments. Microarray and immunoblot analysis results demonstrated that DSAs exposure selectively deregulated senescence associated TGFβR1/FOXO1/p21 signaling. Decrease in β-galactosidase staining further suggested reversion of drug-induced senescence after DSAs exposure. Additionally, DSAs induced profound cell death by activation of apoptotic signaling through bypassing senescence. Furthermore, in vivo and ex vivo imaging analysis demonstrated the tumor targeting behavior of DSAs in mice bearing PC-3 xenograft tumors. The antisenescence and anticancer activity of DSAs was further shown in vivo by inhibiting TGFβR1 proteins and regressing tumor growth through apoptotic induction in the PC-3 xenograft mouse model. Overall, DSAs exhibited such advanced features due to a natural compound in the formulation as a matrix/binder for docetaxel. Overall, DSAs showed superior tumor targeting and improved cellular internalization, promoting docetaxel efficacy. These findings may have great implications in prostate cancer therapy.

Background Cervical cancer (CxCa) ranks as the fourth most prevalent women-related cancer worldwide. Therefore, there is a crucial need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-steroidal, selective estrogen receptor modulator (SERM) that is used as an oral contraceptive in humans. Recent investigations suggest that ORM exhibits potent anti-cancer activity against various types of cancers. Nanoparticulates offer targeted delivery of anti-cancer drugs with minimal toxicity and promise newer approaches for cancer diagnosis and treatment. Therefore, the nanotherapy approach is superior compared to traditional chemotherapy, which is not site-specific and is often associated with various side effects. Methods Pursuing this novel nanotherapy approach, our lab has recently developed ORM-loaded poly [lactic-co-glycolic acid] (PLGA), an FDA-approved biodegradable polymer, nanoparticles to achieve targeted drug delivery and improved bioavailability. Our optimized PLGA-ORM nanoformulation showed improved internalization in both dose- and energy-dependent manners, through endocytosis-mediated pathways in both Caski and SiHa cell lines. Additionally, we employed MTS and colony forming assays to determine the short- and long-term effects of PLGA-ORM on these cells. Results Our results showed that this formulation demonstrated improved inhibition of cellular proliferation and clonogenic potential compared to free ORM. Furthermore, the PLGA-ORM nanoformulation exhibited superior anti-tumor activities in an orthotopic cervical cancer mouse model than free ORM. Conclusion Collectively, our findings suggest that our novel nanoformulation has great potential for repurposing the drug and becoming a novel modality for CxCa management.

The inhibitory kappa B kinases (IKKs) and IKK related kinases are crucial regulators of the pro-inflammatory transcription factor, nuclear factor kappa B (NF-κB). The dysregulation in the activities of these kinases has been reported in several cancer types. These kinases are known to regulate survival, proliferation, invasion, angiogenesis, and metastasis of cancer cells. Thus, IKK and IKK related kinases have emerged as an attractive target for the development of cancer therapeutics. Several IKK inhibitors have been developed, few of which have advanced to the clinic. These inhibitors target IKK either directly or indirectly by modulating the activities of other signaling molecules. Some inhibitors suppress IKK activity by disrupting the protein-protein interaction in the IKK complex. The inhibition of IKK has also been shown to enhance the efficacy of conventional chemotherapeutic agents. Because IKK and NF-κB are the key components of innate immunity, suppressing IKK is associated with the risk of immune suppression. Furthermore, IKK inhibitors may hit other signaling molecules and thus may produce off-target effects. Recent studies suggest that multiple cytoplasmic and nuclear proteins distinct from NF-κB and inhibitory κB are also substrates of IKK. In this review, we discuss the utility of IKK inhibitors for cancer therapy. The limitations associated with the intervention of IKK are also discussed.

Our goal was to design nanocarriers that specifically target and deliver therapeutics to polarized macrophages. Mannose receptors are highly overexpressed on polarized macrophages. In this study, we constructed Pluronic® -F127 polymer and tannic acid (TA) based nanoparticles (F127-TA core nanoparticles) with varying mannose densities. The particle size of the optimized mannose-decorated F127-TA hybrid nanoparticles (MDNPs) was found to be ~ 265 nm with a negative zeta potential of ~ − 4.5 mV. No significant changes in the size and zeta potentials of nanoparticles were observed, which demonstrated structural integrity and stability of the nanoformulation. Physicochemical characteristics of MDNPs were evaluated by FTIR and TGA and demonstrated the presence of mannose units on surface nanoparticles. A mannose-dependent cellular targeting and uptake of MDNPs was found in U937 macrophages. The uptake process was found to vary directly with time and volume of MDNPs nanoparticles. The uptake pattern is higher in M2 than M1. This behavior was also evident from the instantaneous and superior binding profile of M2 macrophage lysate protein with MDNPs over that of M1 macrophage lysate protein. These results demonstrated that an appropriate mannose ligand density was confirmed, suggesting efficient targeting of M2. Altogether, these data support that the MDNPs formulation could serve as a targeted therapeutic guide in the generation of nanomedicine to treat various conditions as an anti-inflammation therapy., Highlights • Mannose receptors are highly overexpressed on polarized macrophages. • The overexpression is attributed to various inflammatory and infection diseases. • This study reports a mannose-decorated hybrid nanoparticle system targeting polarized macrophages. • Hybrid nanoparticles offers biocompatibility and capable to internalize/deliver therapeutics to macrophages.

Bone metastasis occurs in the majority of cancer patients, which hampers quality of life and significantly decreases survival. Aggressive chemotherapy is a traditional treatment regimen that induces severe systemic toxicities. Therefore, bone-directed therapies are highly warranted. We report a novel nanoparticle formulation that is composed of poly(vinylpyrrolidone) and tannic acid core nanoparticles (PVT NPs) that forms self-assembly with zoledronic acid (ZA@PVT NPs). The construction of ZA@PVT NPs was confirmed by particle size, zeta potential, transmission electron microscopy, and spectral analyses. An optimized bone-targeted ZA@PVT NPs formulation showed greater binding and internalization in in vitro with metastasis prostate and breast cancer cells. ZA@PVT NPs were able to deliver ZA more efficiently to tumor cells, which inhibited proliferation of human prostate and breast cancer cells. In addition, ZA@PVT NPs were capable of targeting mouse bones and prostate tumor microarray tissues (ex vivo) while sparing all other vital organs. More importantly, ZA@PVT NPs induce chemo sensitization to docetaxel treatment in cancer cells. Overall, the study results confirm that ZA-based, bone-targeted NPs have great potential for the treatment of bone metastasis in the near future.

Novel strategies modulating the immune system yielded enhanced anticancer responses and improved cancer survival. Nevertheless, the success rate of immunotherapy in cancer treatment has been below expectation(s) due to unpredictable efficacy and off-target effects from systemic dosing of immunotherapeutic(s). As a result, there is an unmet clinical need for improving conventional immunotherapy. Nanotechnology offers several new strategies, multimodality, and multiplex biological targeting advantage to overcome many of these challenges. These efforts enable programming the pharmacodynamics, pharmacokinetics, and delivery of immunomodulatory agents/co-delivery of compounds to prime at the tumor sites for improved therapeutic benefits. This review provides an overview of the design and clinical principles of biomaterials driven nanotechnology and their potential use in personalized nanomedicines, vaccines, localized tumor modulation, and delivery strategies for cancer immunotherapy. In this review, we also summarize the latest highlights and recent advances in combinatorial therapies availed in the treatment of cold and complicated tumors. It also presents key steps and parameters implemented for clinical success. Finally, we analyse, discuss, and provide clinical perspectives on the integrated opportunities of nanotechnology and immunology to achieve synergistic and durable responses in cancer treatment.

Curcumin is an important bioactive component of turmeric and also one of the important natural products, which has been investigated extensively. The precise mode of action of curcumin and its impact on system level protein networks are still not well studied. To identify the curcumin governed regulatory action on protein interaction network (PIN), an interectome was created based on 788 key proteins, extracted from PubMed literatures, and constructed by using STRING and Cytoscape programs. The PIN rewired by curcumin was a scale-free, extremely linked biological system. MCODE plug-in was used for sub-modulization analysis, wherein we identified 25 modules; ClueGo plug-in was used for the pathway’s enrichment analysis, wherein 37 enriched signalling pathways were obtained. Most of them were associated with human diseases groups, particularly carcinogenesis, inflammation, and infectious diseases. Finally, the analysis of topological characteristic like bottleneck, degree, GO term/pathways analysis, bio-kinetics simulation, molecular docking, and dynamics studies were performed for the selection of key regulatory proteins of curcumin-rewired PIN. The current findings deduce a precise molecular mechanism that curcumin might exert in the system. This comprehensive in-silico study will help to understand how curcumin induces its anti-cancerous, anti-inflammatory, and anti-microbial effects in the human body.

Pancreatic cancer (PanCa) is one of the leading causes of death from cancer in the United States. The current standard treatment for pancreatic cancer is gemcitabine, but its success is poor due to the emergence of drug resistance. Natural products have been widely investigated as potential candidates in cancer therapies, and cucurbitacin D (Cuc D) has shown excellent anticancer properties in various models. However, there is no report on the therapeutic effect of Cuc D in PanCa. In the present study, we investigated the effects of the Cuc D on PanCa cells in vitro and in vivo. Cuc D inhibited the viability of PanCa cells in a dose and time dependent manner, as evident by MTS assays. Furthermore, Cuc D treatment suppressed the colony formation, arrest cell cycle, and decreased the invasion and migration of PanCa cells. Notably, our findings suggest that mucin 13 (MUC13) is down-regulated upon Cuc D treatment, as demonstrated by Western blot and qPCR analyses. Furthermore, we report that the treatment with Cuc D restores miR-145 expression in PanCa cells/tissues. Cuc D treatment suppresses the proliferation of gemcitabine resistant PanCa cells and inhibits RRM1/2 expression. Treatment with Cuc D effectively inhibited the growth of xenograft tumors. Taken together, Cuc D could be utilized as a novel therapeutic agents for the treatment/sensitization of PanCa.

Pancreatic tumors are rewired for high-glucose metabolism and typically present with exceptionally poor prognosis. Recently, we have shown that MUC13, which is highly expressed in pancreatic tumors, promotes tumor progression via modulation of HER2 receptor tyrosine kinase activity. Herein, we investigate a novel, MUC13-mediated molecular mechanism responsible for higher glucose metabolism in pancreatic tumors. Our results demonstrate that MUC13 expression leads to the activation/nuclear translocation of NF-κB p65 and phosphorylation of IκB, which in turn upregulates the expression of important proteins (Glut-1, c-Myc, and Bcl-2) that are involved in glucose metabolism. MUC13 functionally interacts and stabilizes Glut-1 to instigate downstream events responsible for higher glucose uptake in pancreatic cancer cells. Altered MUC13 expression by overexpression and knockdown techniques effectively modulated glucose uptake, lactate secretion, and metastatic phenotypes in pancreatic cancer cells. NF-κB inhibitor, Sulfasalazine, abrogates the MUC13 and Glut-1 interaction, and attenuates events associated with MUC13-induced glucose metabolism. Pancreatic ductal adenocarcinoma (PDAC) patient tissue samples also show a positive correlation between the expression of these two proteins. These results delineate how MUC13 rewire aberrant glucose metabolism to enhance aggressiveness of pancreatic cancer and revealed a novel mechanism to develop newer therapeutic strategies for this exceptionally difficult cancer.

MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression through diverse mechanisms. Increasing evidence suggests that miRNA-based therapies, either restoring or repressing miRNA expression and activity, hold great promise. However, the efficient delivery of miRNAs to target tissues is a major challenge in the transition of miRNA therapy to the clinic. Cationic polymers or viral vectors are efficient delivery agents but their systemic toxicity and immunogenicity limit their clinical usage. Efficient targeting and sustained release of miRNAs/anti-miRNAs using nanoparticles (NPs) conjugated with antibodies and/or peptides could reduce the required therapeutic dosage while minimizing systemic and cellular toxicity. Given their importance in clinical oncology, here we focus on the development of miRNA nanoformulations to achieve enhanced cellular uptake, bioavailability, and accumulation at the tumor site.

Background The management of pancreatic cancer (PanCa) is exceptionally difficult due to poor response to available therapeutic modalities. Tubulins play a major role in cell dynamics, thus are important molecular targets for cancer therapy. Among various tubulins, βIII and βIV-tubulin isoforms have been primarily implicated in PanCa progression, metastasis and chemo-resistance. However, specific inhibitors of these isoforms that have potent anti-cancer activity with low toxicity are not readily available. Methods We determined anti-cancer molecular mechanisms and therapeutic efficacy of a novel small molecule inhibitor (VERU-111) using in vitro (MTS, wound healing, Boyden chamber and real-time xCELLigence assays) and in vivo (xenograft studies) models of PanCa. The effects of VERU-111 treatment on the expression of β-tubulin isoforms, apoptosis, cancer markers and microRNAs were determined by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses. Results We have identified a novel small molecule inhibitor (VERU-111), which preferentially represses clinically important, βIII and βIV tubulin isoforms via restoring the expression of miR-200c. As a result, VERU-111 efficiently inhibited tumorigenic and metastatic characteristics of PanCa cells. VERU-111 arrested the cell cycle in the G2/M phase and induced apoptosis in PanCa cell lines via modulation of cell cycle regulatory (Cdc2, Cdc25c, and Cyclin B1) and apoptosis - associated (Bax, Bad, Bcl-2, and Bcl-xl) proteins. VERU-111 treatment also inhibited tumor growth (P

Background: Pancreatic cancer (PC) is a disease with poor prognosis and survival rate. There is a pertinent need to identify the risk factors of this disease. The purpose of this study is to use machine learning methods to identify a subset of factors (a.k.a. features) from the PLCO dataset as predictors of PC. The Prostate, Lung, Colorectal and Ovarian (PLCO) cancer dataset is collected by the National Cancer Institute from 155,000 participants (49.5% male). Each participant responded to three questionnaires consisting of 65 questions about demographics, illness history, and family background. Method: This is an optimal feature selection problem. The goal is to identify the subset of features that predict PC with highest probability. There are two challenges to solving this problem: (1) the problem is computationally intractable (there are n65 possible subsets of features where n is the number of values each feature can take on average), and (2) the PLCO dataset is highly imbalanced (only 0.48% participants have PC). The dataset was balanced by downsampling the majority class. Eleven methods were used for feature selection. Classification was done by 25 classifiers using the selected features from each of the 11 methods, thereby generating 11 × 25=275 results. All methods used for balancing, feature selection and classification are well-established in the field of machine learning. For each classifier, the baseline was obtained by classifying the balanced datasets using all features. The dataset was used 60% for training and 40% for testing. Hyperparameters were estimated via cross-validation on the training set. Results: Approximately 11% of the 275 classification results were accurate which were distributed across the different balancing, feature selection and classification methods. Among the 65 features, 17 were chosen by more than 50% of the feature selection methods. Among them, race, occupation (retired or not, indicative of age), smoking, prior history of any cancer, and number of relatives with PC were more discriminative than the others. Considering a subset of two features for male participants, probability of PC given age when told had inflamed prostate is 70+ and number of cigarettes smoked daily is 80+ was the highest (0.032) followed by age when told had inflamed prostate is 70+ and prior history of any cancer (0.03). For females, probability of PC given number of relatives with PC is 2+ and number of cigarettes smoked daily is 61-80 was the highest (0.156) followed by number of relatives with PC is 2+ and number of tubal/ectopic pregnancies is 2+ (0.137). Conclusions: The study found that age, smoking, prior history of cancer and relatives with cancer are the prominent risk factors of PC. Inflamed prostrate for males and tubal/ectopic pregnancies for females are also risk factors of PC. When two of these factors occur in conjunction, the risk of PC may increase even more. However, that is not necessarily the case when three or more of these factors occur in conjunction. Citation Format: Ananya Dutta, Bonny Banerjee, Sheema Khan, Subhash Chauhan. Using machine learning to identify the risk factors of pancreatic cancer from the PLCO dataset [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-073.

microRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of their target mRNAs post transcriptionally. miRNAs are known to regulate not just a gene but the whole gene network (signaling pathways). Accumulating evidence(s) suggests that miRNAs can work either as oncogenes or tumor suppressors, but some miRNAs have a dual nature since they can act as both. miRNA 205 (miR-205) is one such highly conserved miRNA that can act as both, oncomiRNA and tumor suppressor. However, most reports confirm its emerging role as a tumor suppressor in many cancers. This review focuses on the downregulated expression of miR-205 and discusses its dysregulation in breast, prostate, skin, liver, gliomas, pancreatic, colorectal and renal cancers. This review also confers its role in tumor initiation, progression, cell proliferation, epithelial to mesenchymal transition, and tumor metastasis. Restoration of miR-205 makes cells more sensitive to drug treatments and mitigates drug resistance. Additionally, the importance of miR-205 in chemosensitization and its utilization as potential biomedicine and nanotherapy is described. Together, this review research article sheds a light on its application as a diagnostic and therapeutic marker, and as a biomedicine in cancer.

Background Pancreatic cancer is one of the deadliest malignancies, and fourth leading cause of cancer-related deaths in the United States. Aberrant glucose metabolism in cancer cells leads to enhanced tumor growth and aggressiveness. Herein, we have identified an important constituent, steviol from the plant Stevia rebaudiana, which inhibits pancreatic cancer cells via modulation of glucose metabolism and protein translation. Methods Glucose and Lactate assays were performed in pancreatic cancer cells (HPAF-II and AsPC1) using kits purchased from Cayman Chemical. The effect of steviol on the growth, colonization and invasion of pancreatic cancer cells was determined by using in vitro functional assays for cell viability (MTT and xCELLigence), migration (Boyden chambers), invasion (Matrigel) and colonogenicity. The cell cycle study was performed using propidium iodide and analysis done through ModFit software. Immunoblotting was performed to analyze the expression of key proteins associated with translation machinery and apoptotic signaling pathways. Results Our results demonstrate that steviol inhibits proliferation and invasion of pancreatic cancer cells and modulates their glucose uptake and lactate secretion. Steviol treatment regulates the expression of proteins that control the translation machinery in a cell, which include mTOR, 4EBP1, eIF4E, eIF4B and eIF4G. Treatment of cells with steviol was observed to modulate cell cycle regulatory proteins, including p18, p21 and Cyclin D1 and block the cell cycle progression at G1 phase This followed the activation of apoptosis as indicated by upregulation of apoptotic protein, Bax and PARP cleavage. Conclusion: These results suggest that steviol suppresses pancreatic cancer growth and progression by regulating the glucose metabolism and protein translation. This prospects steviol as a promising therapeutic modality for pancreatic cancer prevention and treatment alone or in combination with other existing chemotherapeutic drugs. Citation Format: Sonam Kumari, Sheema Khan, Mohammed Sikander, Shabnam Malik, Subhash C. Chauhan, Meena Jaggi. Steviol induces pancreatic cancer cell death via targeting glucose metabolism and protein translation pathways [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 4195.

Background: Colorectal carcinoma (CRC) is the second leading cause of cancer related deaths in the United States. The five-year survival rate of patients diagnosed with distant stages declines to 14%, which is of concern when compared to 90% for localized-stage and 71% for regional stage disease. This necessitates the need for early diagnostic biomarkers, to minimize poor drug response/resistance, recurrence, metastasis and mortality. Disproportionate biochemical stressors increase the risk of developing CRC and its progression by influencing molecular drivers within the coding and noncoding parts of the genome. Thus, understanding the biological mechanism of these stress factors on the molecular drivers of this disease can provide pivotal information pertinent to CRC development and progression. Recently, our laboratory has identified a novel long noncoding RNA (lncRNA) namely, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which is highly over-expressed in CRC and is involved in its pathogenesis and is regulated by transcription factor Nuclear Factor of Activated T cell 1 (NFATc1). Methods: Archived human CRC tissues were stained using a recently standardized Z-probe technology. TCGA database of ~600 CRC patients was also analyzed using the bioinformatic approach. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral constructs were used to generate stable lncRNA MALAT1 expressing cell lines. CRISPR/Cas9 constructs were used to knockdown lncRNA MALAT1 and NFATc1. Mouse model was used to verify the stress induced expression of MALAT1 and NFATc1. ReCLP (Reversible Cross-Linked Precipitation) and iRAP (invitro RNA Antisense Proteomics) studies are in progress to identify the associated proteins and complexes. Results: RNAScope analysis showed MALAT1 to be highly over-expressed in human CRC tissues. MALAT1 expression increased with stage and negatively correlated with the tumor size. TCGA database analysis confirmed our findings. Multiple NFATc1 binding site were identified by ChIPseq database. Overexpression of transcription factor NFATc1 upregulated lncRNA MALAT1 expression. CRISPR/Cas9 based knockdown of NFATc1 downregulated NFATC1 and lncRNA MALAT1, but vice versa was not true, indicating NFATc1 to be upstream of lncRNA MALAT1. Also, expression of MALAT1, NFATc1 and IL-6 were highly upregulated by biochemical stressor cortisol in mouse colonic tissues. Further studies are in progress for direct association of NFATc1 on MALAT1 promoter and mechanism by which the stress factor regulate NFATc1 expression and hence lncRNA MALAT1 expression. Conclusion: This study helps to understand influence of biochemical stress factors on long noncoding RNA MALAT1 and transcription factor NFATc1 etiology. Early diagnosis of these molecular markers will help in designing novel preventive/therapeutic strategies to reduce CRC progression, metastasis and hence mortality. Citation Format: Kyle Doxtater, Chidi Zacheaus, Radhika Sekhri, Utkarsh K. Mishra, Zachary E. Stiles, Nitish Mishra, Chittibabu Guda, Nadeem Zafar, Mahul Amin, Pradeep Shukla, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan, Manish K. Tripathi. Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis [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 3142.

The United States Food and Drug Administration has permitted number of therapeutic agents for cancer treatment. Most of them are expensive and have some degree of systemic toxicity which makes overbearing in clinical settings. Although advanced research continuously applied in cancer therapeutics, but drug resistance, metastasis, and recurrence remain unanswerable. These accounts to an urgent clinical need to discover natural compounds with precisely safe and highly efficient for the cancer prevention and cancer therapy. Gambogic acid (GA) is the principle bioactive and caged xanthone component, a brownish gamboge resin secreted from the of Garcinia hanburyi tree. This molecule showed a spectrum of biological and clinical benefits against various cancers. In this review, we document distinct biological characteristics of GA as a novel anti-cancer agent. This review also delineates specific molecular mechanism(s) of GA that are involved in anti-cancer, anti-metastasis, anti-angiogenesis, and chemo-/radiation sensitizer activities. Furthermore, recent evidence, development, and implementation of various nanoformulations of gambogic acid (nanomedicine) have been described.

Formalin-fixed paraffin embedded (FFPE) tissues are a valuable resource for biomarker discovery in order to understand the etiology of different cancers and many other diseases. Proteins are the biomarkers of interest with respect to FFPE tissues as RNA degradation is the major challenge in these tissue samples. Recently, non-protein coding transcripts, long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. RNA sequencing (RNA-seq) or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are the only validated methods to evaluate and study lncRNA expression and neither of them provides visual representation as immunohistochemistry (IHC) provides for proteins. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to visually identify and quantify lncRNA in FFPE tissues. This assay is highly sensitive and identifies transcripts visible within different cell types and tumors. We have detected a scarcely expressed tumor suppressor lncRNA NRON (non-coding repressor of nuclear factor of activated T-cells (NFAT)), a moderately expressed oncogenic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. Additionally, we have observed an increase in MALAT1 expression in different stages of colorectal cancer.

The therapeutic application of microRNA(s) in the field of cancer has generated significant attention in research. Previous studies have shown that miR-205 negatively regulates prostate cancer cell proliferation, metastasis, and drug resistance. However, the delivery of miR-205 is an unmet clinical need. Thus, the development of a viable nanoparticle platform to deliver miR-205 is highly sought. A novel magnetic nanoparticle (MNP)-based nanoplatform composed of an iron oxide core with poly(ethyleneimine)-poly(ethylene glycol) layer(s) was developed. An optimized nanoplatform composition was confirmed by examining the binding profiles of MNPs with miR-205 using agarose gel and fluorescence methods. The novel formulation was applied to prostate cancer cells for evaluating cellular uptake, miR-205 delivery, and anticancer, antimetastasis, and chemosensitization potentials against docetaxel treatment. The improved uptake and efficacy of formulations were studied with confocal imaging, flow cytometry, proliferation, clonogenicity, Western blot, q-RT-PCR, and chemosensitization assays. Our findings demonstrated that the miR-205 nanoplatform induces significant apoptosis and enhancing chemotherapeutic effects in prostate cancer cells. Overall, these study results provide a strong proof-of-concept for a novel nonviral-based nanoparticle protocol for effective microRNA delivery to prostate cancer cells.

The quantitative measurement of structural alterations at the nanoscale level is important for understanding the physical state of biological samples. Studies have shown that the progression of cancer is associated with the rearrangements of building blocks of cells/tissues such as DNA, RNA, lipids, etc. Partial wave spectroscopy is a recently developed mesoscopic physics-based spectroscopic imaging technique which can detect such nanoscale changes in cells/tissues. At present, chemotherapy drug treatment is the only effective form of treatment; however, the development of drug-resistant cancer cells is a major challenge for this treatment. Earlier PWS analyses of prostate cancer cells, a 2D structure, have shown that drug-resistant cancer cells have a higher degree of structural disorder compared to drug-sensitive cancer cells. At the same time, structural properties of the metastasize tumor grown to 3D structure from drug-resistant and drug-sensitive cancer cells within the body is not well studied. In this paper, the structural properties of tissues from grown 3D tumors, generated from docetaxel drug-sensitive and drug-resistant prostate cancer cells xenografted into a mouse model, are studied. The results show that xenografted tumor tissues from drug-resistant cells have higher disorder strength than the tumor generated from drug-sensitive prostate cancer cells. Potential applications of the technique to assess chemotherapy effectiveness in cancer treatment are discussed., 5 figures, 13 pages

BACKGROUND: Poor prognosis of pancreatic cancer (PanCa) is associated with lack of an effective early diagnostic biomarker. This study elucidates significance of MUC13, as a diagnostic/prognostic marker of PanCa. METHODS: MUC13 was assessed in tissues using our in-house generated anti-MUC13 mouse monoclonal antibody and analyzed for clinical correlation by immunohistochemistry, immunoblotting, RT-PCR, computational and submicron scale mass-density fluctuation analyses, ROC and Kaplan Meir curve analyses. RESULTS: MUC13 expression was detected in 100% pancreatic intraepithelial neoplasia (PanIN) lesions (Mean composite score: MCS=5.8; AUC >0.8, P0.8; P

Ovarian cancer continues to be a leading cause of cancer related deaths for women. Anticancer agents effective against chemo-resistant cells are greatly needed for ovarian cancer treatment. Repurposing drugs currently in human use is an attractive strategy for developing novel cancer treatments with expedited translation into clinical trials. Therefore, we examined whether ormeloxifene (ORM), a non-steroidal Selective Estrogen Receptor Modulator (SERM) currently used for contraception, is therapeutically effective at inhibiting ovarian cancer growth. We report that ORM treatment inhibits cell growth and induces apoptosis in ovarian cancer cell lines, including cell lines resistant to cisplatin. Furthermore, ORM treatment decreases Akt phosphorylation, increases p53 phosphorylation, and modulates the expression and localization patterns of p27, cyclin E, cyclin D1, and CDK2. In a pre-clinical xenograft mouse ORM treatment significantly reduces tumorigenesis and metastasis. These results indicate that ORM effectively inhibits the growth of cisplatin resistant ovarian cancer cells. ORM is currently in human use and has an established record of patient safety. Our encouraging in vitro and pre-clinical in vivo findings indicate that ORM is a promising candidate for the treatment of ovarian cancer.

The biomechanical properties of cells and tissues may be instrumental in increasing our understanding of cellular behavior and cellular manifestations of diseases such as cancer. Nanomechanical properties can offer clinical translation of therapies beyond what are currently employed. Nanomechanical properties, often measured by nanoindentation methods using atomic force microscopy, may identify morphological variations, cellular binding forces, and surface adhesion behaviors that efficiently differentiate normal cells and cancer cells. The aim of this review is to examine current research involving the general use of atomic force microscopy/nanoindentation in measuring cellular nanomechanics; various factors and instrumental conditions that influence the nanomechanical properties of cells; and implementation of nanoindentation methods to distinguish cancer cells from normal cells or tissues. Applying these fundamental nanomechanical properties to current discoveries in clinical treatment may result in greater efficiency in diagnosis, treatment, and prevention of cancer, which ultimately can change the lives of patients.

In this study, we for the first time, investigated the potential anti-cancer effects of a novel analogue of cucurbitacin (Cucurbitacin D) against cervical cancer in vitro and in vivo. Cucurbitacin D inhibited viability and growth of cervical cancer cells (CaSki and SiHa) in a dose-dependent manner. IC50 of Cucurbitacin D was recorded at 400 nM and 250 nM in CaSki and SiHa cells, respectively. Induction of apoptosis was observed in Cucurbitacin D treated cervical cancer cells as measured by enhanced Annexin V staining and cleavage in PARP protein. Cucurbitacin D treatment of cervical cancer cells arrested the cell cycle in G1/S phase, inhibited constitutive expression of E6, Cyclin D1, CDK4, pRb, and Rb and induced the protein levels of p21 and p27. Cucurbitacin D also inhibited phosphorylation of STAT3 at Ser727 and Tyr705 residues as well as its downstream target genes c-Myc, and MMP9. Cucurbitacin D enhanced the expression of tumor suppressor microRNAs (miR-145, miRNA-143, and miRNA34a) in cervical cancer cells. Cucurbitacin D treatment (1 mg/kg body weight) effectively inhibited growth of cervical cancer cells derived orthotopic xenograft tumors in athymic nude mice. These results demonstrate the potential therapeutic efficacy of Cucurbitacin D against cervical cancer.

Background Lung cancer is the leading cause of cancer deaths across globe. Statistically, 70-80% of lung cancer cases fall under non-small cells lung cancer (NSCLC) category. Recently, usage of natural compounds (e.g. EGCG, curcumin, resveratrol, apigenin, etc.,) for cancer therapy has gained lots of attention. However, poor dissolution profiles of these natural compounds lead to unfavorable pharmacological doses and administration. For this purpose, we have candidate, a water-soluble natural polyphenol, Tannic acid (TA, C76H52O46), which, a natural polyphenol that is richly found in plants such as green tea. This molecule exhibits anti-oxidant and anti-proliferative activities, which is being used as an alternative therapeutic option in cancer therapy. However, role of TA in NSCLC was not examined. Therefore, the aim of present study is to assess the molecular effects of TA in NSCLC cells. Methods For this study, two NSCLC cell lines (A549 and H1299) and one normal lung cell line (BEAS-2B) were used. We investigated the anti-proliferative effects of TA on NSCLC cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT), and colony formation assays. The anti-invasiveness and anti-migratory potential of TA was evaluated through Matrigel and Boyden chamber studies, respectively. Cell cycle and apoptotic effects of TA was evaluated through flow cytometry and western blot studies. The quantification of secreted VEGF was assessed by ELISA. Also, for affirmation results of gene expression profiles we performed q-PCR studies. Results Cell proliferation study demonstrated a dose dependent anti-cancer effects of TA on NSCLC cells. However, TA had no significant toxicity effects on human bronchial epithelial cells. Similarly, colony formation results exhibited dose dependence reduction in response to escalated dose of TA exposure. Both the invasion and migration results confirm its anti-metastatic role. Extracellular VEGF quantification showed lower VEGF secretion with TA treatments compared to the control cells. The cell cycle analysis evidently showed TA efficiently induced G1 phase cell arrest in NSCLC cells which was further confirmed through western blotting studies of relevant proteins (Cyclin D1, P53, p21, p18, BAX, BCL-2). We also achieved to show the decreased expression of VEGFR2, p-AKT, and cleaved caspase 7 indicating TA induces apoptosis. Conclusion Overall, these results demonstrate illustrate anti-cancer activity of TA by targeting the VEGF related pathways in NSCLC. This approach can lead to an alternative therapeutic strategy for lung cancer, with ideal PK/PD profiles. Citation Format: Elham Hatami, Prashanth K.B. Nagesh, Pallabita Chowdhury, Advit Bhaskar Shetty, Manish K. Tripathi, Subhash Chauhan, Meena Jaggi, Murali Yallapu. Tannic acid: A natural anticancer agent for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1871.

Purpose: Prostate cancer (PrCa) is one of the leading causes of cancer-related deaths among men in the United States. Chemotherapy with docetaxel holds great promise and important therapeutic option for PrCa treatment. However, chemotherapy is often associated with incomplete cell death and leads to persistent senescent phenotype. Such phenotypic cancer cells survive and promotes tumor development, recurrence, and drug resistance. Altogether, targeting these cells can enhance chemotherapy outcomes. In the current study, we established a tannic acid-docetaxel scaffold nanoparticles (TDS NP) platform that allows for facile targeting and inhibition of drug induced senescence in prostate cancer. Methods: TDS NP were prepared by solvent evaporation and extrusion process using a series of tannic acid and docetaxel weight ratios. The scaffold self-assembly formation was determined using a steady-state fluorescence quenching, FT-IR, XRD, DSC, and TGA. The surface and morphological properties of TDS NPs were determined by dynamic light scattering and transmission electron microscopy. The biocompatibility assessment was characterized by hemolysis assay. C4-2 and PC-3 cell lines were used as PrCa model systems for in vitro and in vivo studies. The cellular internalization (in vitro) and accumulation (in vivo) was examined using dye-tagged TDS NPs. The superior in vitro anti-cancer and metastatic potential of TDS NPs were evaluated using proliferation, colony formation, migration, invasion, and immunoblotting assays. The anti-senescence ability of TDS NPs was examined through β-Galactosidase Staining Kit. A PC-3 xenograft mouse model was used to examine its superior therapeutic activity over free docetaxel treatment. Results: A series of physico-chemical analyses confirmed the integrity of TDS NP. An optimized formulation exhibited a spherical shape particle formulation with 124.13 ± 2.16 nm with a negative zeta potential of -14.0 mV. TDS NPs formulation exhibited superior internalization capacity in PrCa cells in a dose- and time-dependent manner. In vitro functional studies confirm superior therapeutic activity of TDS NPs over free docetaxel treatment. Enhanced pro-apoptotic while downregulating anti-apoptotic effects with treatment of TDS NPs in comparison to native drug. A profound inhibition of β-galactosidase activity was noticed with TDS NPs treatment. In vivobiodistribution studies confirm efficient accumulation of TDS NPs in mice. In addition, TDS NPs showed robust antitumor activity against PC-3 xenografts. Such improved activity was correlated with increased inhibition of senescence and induced apoptosis. Conclusion: In summary, we developed a novel tannic acid-docetaxel scaffold nanoparticle formulation that is capable of inhibiting senescence, minimizing drug resistance, and inducing apoptosis in prostate cancer. Citation Format: Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Elham Hatami, Sonam Kumari, Vivek Kumar Kashyap, Bilal Hafeez, Sheema Khan, Manish Kumar Tripathi, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Tannic acid-docetaxel scaffold nanoparticles for improved prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3634.

Colorectal Cancer(CRC) is one of the leading cause of cancer related death in the United States today in both men and women. With an expected 50,260 deaths in 2017 it is the second leading cause of cancer related deaths among men and the third among women in the United states. Often in colorectal cancer it is not the primary tumor that is the killer, but it is the metastatic sites that develop in the later stages of the disease that is the true killer. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. What is metastasis? Metastasis is the transfer of disease from the primary tumor site to another unconnected site within the body through a process called the metastatic cascade. Metastasis develops slowly over time, due to it being an inefficient process having to overcome several natural defenses within the body. One of these defenses is Anoikis, cell death due to lack of attachment to the extracellular matrix. The development of Anoikis resistant is key in the process of metastasis, because of the need of the cells to detach and circulate the body before settling at a new site within the body. The inhibition of Anoikis resistance development in CRC could lead to improve patient outcome and the failure of metastasis to develop in the lifetime of the disease in the patient. Thus, characterizing and developing methods of inhibition of Anoikis resistance could lead to the decrease in cancer related deaths in the United States as well as finding effect therapeutic options. One of the key characteristics of Anoikis resistance in cells is the develop of stem cell like characteristics, and expression of key stemness factors of Sox2, Oct4, and NANOG. The expression of these factors allows for the cells to overcome their need to be attached to the ECM and travel through the blood stream. We have found through analysis that the membrane bound muicin MUC13 interacts with Sox2 increasing cell stemness leading to the development of anoikis resistance in Colorectal Cancer. Cell cycle analysis of Sw620 cells with high endogenous MUC13 expression showed an 80% less cell death when compare to isogenic Sw480 cells after 48 hours. Western blot anaylsis of the time points 0,24,36, and 48 hrs showed an increase in Sox2 expression and MUC13 expression in Sw620 cells during 24 and 36hrs. To further understand this relationship MUC13 knockdown and overexpressing cells lines will be analyzed to see if any there is a change in the Anoikis phenotype of Sw620 and Sw480 cells. This will also be done with Sox2 knockdown and overexpression. We will also test different therapeutic options, specifically a novel non-viral delivery of MUC13 Crispr Cas9 plasmid to see it effect on Anoikis resistances. Citation Format: Kyle Doxtater, Chidi Zacheaus, Fatemeh Keramatnia, Mehdi Chaib, Manish Triparthi, Subhash Chauhan. MUC13 interacts with SOX2 leading to cell stemness and Anoikis resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2647.

Background: Cervical cancer (CxC) is a leading cause of mortality and morbidity among women worldwide. Current chemotherapeutic agents for CxC have shown systemic toxicity in CxC patients. Ormeloxifene (ORM) is a non-toxic and non-steroidal drug with well-defined pharmacokinetic and pharmacodynamic properties in humans. Studies have shown its anti-cancer potential in various pre-clinical mouse models. Here, we have synthesized and characterized a novel analogue of ormeloxifene, Bromo-ormeloxifene (Br-ORM), which showed more therapeutic efficacy against CxC in vitro and in vivo model systems. Methodology: The effect of Br-ORM on CxC cells (CaSki and SiHa) growth and proliferation was determined by colony formation and MTS assays. Molecular docking of Br-ORM with β-catenin was done by AutoDock4 software. Effect of Br-ORM on the expression of epithelial-to mesenchymal (EMT) markers (N-cadherin, slug, snail), MMPs (MMP2 and MMP3) and miR-200a was analyzed by Western blot and qPCR analyses respectively. Apoptosis analysis was done by Annexin-V staining kit. Effect of Br-ORM on β-catenin cellular localization in CxC cells was analyzed by immunofluorescence analysis. The anti-tumor efficacy of Br-ORM was investigated in orthotopic xenograft mouse model of CxC. Results: Br-ORM (10-20 µM) effectively inhibited growth and proliferation of CxC cells in a dose and time-dependent manner as compared to ORM. Br-ORM efficiently suppressed metastatic phenotypes of CxC cells as determined by significant (P Conclusion: These results suggest that Br-ORM inhibits the metastatic phenotypes of CxC cells via targeting β-catenin signaling pathway. Br-ORM could be used as a novel therapeutic modality for the treatment of CxC. Citation Format: Mohammed Sikander, Shabnam Malik, Bilal B. Hafeez, Sonam Kumari, Sheema Khan, John Apraku, Hassan Mandil, Andrew E. Massey, Aditya Ganju, Parvez Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Bromo-ormeloxifene inhibits epithelial mesenchymal transition via targeting β-catenin signaling pathways in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4809.

Background: Although, surgical resection and chemotherapy are the gold standard for treating pancreatic cancer (PanCa), poor patient survival remains the problem. Despite being one of the most common oncogenes in human cancer, to date, no success has been achieved to inhibit KRAS. Targeting KRAS has been shown to synergize anti-cancer activity of gemcitabine in pancreatic cancer. Herein, we have developed a supermagnetic iron oxide (SP) nanoparticles for the sustained delivery of KRAS siRNA to the tumor and simultaneous sensitization of gemcitabine in PanCa. Methods: PanCa cells, AsPC1 and Panc-1 were used in the study. A precipitation approach was employed to develop the SP formulation. These particles were further conjugated with siKRAS (12D; most common KRAS mutation) and investigated for its anticancer efficacy alone and in combination with gemcitabine. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatiblity (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. kRAS silencing was confirmed at both mRNA and protein levels using quantitative reverse-transcription PCR and Western blotting, respectively. Anti-cancer efficacy of SP-siKRAS particles alone or in combination with gemcitabine treatment was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), colonogenicity and tumor spheroid formation. Results: Our results demonstrate optimal particle size (190.137 nm) and zeta potential (18.73mV) of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PanCa cells and suppressed KRAS G12D expression as well as its downstream targets, YAP and PDL-1. SP-siKRAS improved gemcitabine response as observed through enhanced inhibition of cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. Additionally, SP-siKRAS together with gemcitabine resulted in the activation of death related mechanisms in PanCa cells, such as Bax, bcl-2, PARP cleavage. Interestingly, SP-siKRAS inhibited the secondary tumorsphere formation in combined PanCa and cancer associated fibroblast (CAFs) cells, alone as well as in combination with gemcitabine. At day 14, analysis of secondary tumorspheres, treated with SP-siKRAS revealed diminished levels of KRAS G12D through PCR. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited haemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body. Conclusion: Therefore, SP-siKRAS provide a highly efficient and safe platform for silencing KRAS and improving gemcitabine therapy in pancreatic cancer. Citation Format: Nirnoy Dan, Sheema Khan, Saini Setua, Sonam Kumari, Pallabita Chowdhury, Kamalika Samanta, Meena Jaggi, Murali Yallappu, Subhash Chauhan. Nanodelivery platform for targeting mutant-KRAS and improving response to gemcitabine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3625.

Background-Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is no substantial progress in cancer therapeutics due to barrier against intracellular drug delivery. Our group has previously identified that microRNA-145 (miR-145) is downregulated in PanCa, the restoration of which inhibits tumor growth and enhances gemcitabine sensitivity. However, lack of an effective tumor-specific delivery system remains an unmet clinical challenge for successful translation of microRNAs. Herein, we have utilized exosomes (extra cellular vesicles) isolated from pancreatic cancer cells as a vehicle for delivering miR-145 in PanCa cells. Exosomes, unlike other vectors for gene delivery, are non-immunogenic in nature and can protect the RNA/gene of interest from digestion making them a more efficient vehicle for gene delivery. Method- Exosomes were isolated from pancreatic cancer cells HPAF-II, AsPC-1 by the principle of precipitation using exosome isolation reagents. Physico-chemical characterization (DLS, TEM), presence of exosomal marker (CD63; immunoblotting), protein concentration (Bradford assay) and cellular internalization (confocal microscopy) of the exosomes were performed. miR-145 transfection was performed in pancreatic cancer cells using exofectamine. miR-145 restitution and anti-cancer efficacy was investigated using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), colonogenicity and tumor spheroid formation. The effect of exosome mediated miR-145 restitution was investigated using Western blotting and PCR analysis. Results- Exosomes show effective size and zeta potential (AsPC-1:164 nm -9.4mV respectively; HPAF-II: 157 nm -9.2 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed by analyzing the expression of exosomal markers, such as CD63. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. miR-145 loaded exosome treatment efficiently delivered miR-145 into the cancer cells and inhibited the tumorigenic features such as, proliferation, migration, invasion and colonogenicity of PanCa cells. The restoration of lost miR-145 levels using exosome mediated delivery was confirmed in PanCa cells using PCR analysis. MicroRNA-145 fold expression was found to be high at both pH, 7.4 and 6.8, which further indicates relevance for their utilization in the delivery of therapeutic modalities. Conclusion- Our observations offer importance of the utilization of exosomes for delivery of therapeutic modalities and developing personalized medicine in PanCa. Citation Format: Kamalika Samanta, Sheema Khan, Saini Setua, Sonam Kumari, Nirnoy Dan, Murali Mohan Yallapu, Meena Jaggi, Subhash Chandra Chauhan. MicroRNA-145 gene therapy for pancreatic cancer using exosome as carrier source derived from cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3564.

Background: Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, developing novel therapeutic strategies will help for the management of this disease. We recently identified VERU-111, a novel synthetic molecule which showed potent anti-cancer effect against PanCa via targeting clinically important βIII and βIV tubulin isoforms. In this study, we synthesized and characterized its novel nanoformulation (MNP-VERU) and evaluated its therapeutic effects in vitro and xenograft mouse model. Methods: MNPs were prepared by chemical precipitation method and loaded with VERU-111 using diffusion method. This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). The internalization of MNP-VERU was achieved after 6 hours incubation with MNP-VERU in PanCa cells. To determine therapeutic efficacy of MNP-VERU, we performed various in vitro (MTS, wound healing, boyden chamber real-time xCELLigence, and apoptosis assays) and in vivo (mouse tumor xenograft) studies using PanCa. Effect of MNP-VERU on various key oncogenic signaling pathways, and miRNAs was evaluated by Western blot, immunohistochemistry (IHC), confocal microscopy, qRT-PCR and in situ hybridization (ISH) analyses respectively. Result: Our novel MNP-VERU formulation provided average size of 110 nm in dynamic light scattering (DLS) and exhibited -8.23 to -11.65 mV zeta potential with an outstanding loading efficiency (94%). Cellular uptake and internalization studies demonstrate that MNP-VERU escape lysosomal degradation, providing efficient endosomal release to cytosol. MNP-VERU showed remarkable anti-cancer potential in various PanCa cells (Panc-1, AsPC-1, HPAF-II, BxPC-3, MiaPaca) and more effectively repressed βIII and βIV tubulin isoforms via restoring the expression of miR-200c. MNP-VERU more effectively suppressed AsPC-1 cells derived xenograft tumors in athymic nude mice. Conclusions: Taken together, our results suggest that MNP-VERU has more anti-cancer potential than free VERU-111 against PanCa. MNP-VERU may reduce the toxicity and improve the bioavailability of free VERU-111 and could be used for the management of PanCa. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghui Wang, Neeraj Chauhan, Prashanth K. Nagesh, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan. Nanoparticle formulation of VERU 111 for pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3619.

Cancer is the second leading cause of death in the United States. Conventional therapies cause widespread systemic toxicity and lead to serious side effects which prohibit their long term use. Additionally, in many circumstances tumor resistance and recurrence is commonly observed. Therefore, there is an urgent need to identify suitable anticancer therapies that are highly precise with minimal side effects. Curcumin is a natural polyphenol molecule derived from the Curcuma longa plant which exhibits anticancer, chemo-preventive, chemo- and radio-sensitization properties. Curcumin’s widespread availability, safety, low cost and multiple cancer fighting functions justify its development as a drug for cancer treatment. However, various basic and clinical studies elucidate curcumin’s limited efficacy due to its low solubility, high rate of metabolism, poor bioavailability and pharmacokinetics. A growing list of nanomedicine(s) using first line therapeutic drugs have been approved or are under consideration by the Food and Drug Administration (FDA) to improve human health. These nanotechnology strategies may help to overcome challenges and ease the translation of curcumin from bench to clinical application. Prominent research is reviewed which shows that advanced drug delivery of curcumin (curcumin nanoformulations or curcumin nanomedicine) is able to leverage therapeutic benefits by improving bioavailability and pharmacokinetics which in turn improves binding, internalization and targeting of tumor(s). Outcomes using these novel drug delivery systems have been discussed in detail. This review also describes the tumor-specific drug delivery system(s) that can be highly effective in destroying tumors. Such new approaches are expected to lead to clinical trials and to improve cancer therapeutics.

Prostate cancer (PC) is the most frequently diagnosed disease in men in the United States. Curcumin (CUR), a natural polyphenol, has shown potent anti-cancer efficacy in various types of cancers. However, suboptimal pharmacokinetics and poor bioavailability limit its effective use in cancer therapeutics. Several successful CUR nanoformulations have recently been reported which improve upon these features; however there is no personalized safe nanoformulation for prostate cancer. This study contributes two important scientific aspects of prostate cancer therapeutics. The first objective was to investigate the comparative cellular uptake and cytotoxicity evaluation of β-cyclodextrin (CD), hydroxyl propyl methyl cellulose (cellulose), poly(lactic-co-glycolic acid) (PLGA), magnetic nanoparticles (MNP), and dendrimer based CUR nanoformulations in prostate cancer cells. Curcumin loaded cellulose nanoparticles (cellulose-CUR) formulation exhibited the highest cellular uptake and caused maximum ultrastructural changes related to apoptosis (presence of vacuoles) in prostate cancer cells. Secondly, the anti-cancer potential of the optimized cellulose-CUR formulation was evaluated in cell culture models using cell proliferation, colony formation and apoptosis (7-AAD staining) assays. In these assays, the cellulose-CUR formulation showed improved anti-cancer efficacy compared to free curcumin. Our study shows, for the first time, the feasibility of cellulose-CUR formulation and its potential use in prostate cancer therapy.

Objective: Pancreatic cancer (PanCa) is the third deadliest cancer in United States with a poor survival rate. Despite extensive research efforts, there is not any substantial progress in cancer therapeutics due to impediments against intracellular drug delivery. Thus, novel delivery vehicles are required that are biocompatible and non-immunogenic. This is possible by utilizing an autologous biological material as delivery vehicles that can be applied as a personalized medicine. Towards this, our lab has optimized an exosomes based therapeutic approach, which utilizes exosomes isolated from the cultured tumor adjacent normal (NAT) fibroblast cells. We utilized this scaffold for safe and effective delivery of therapeutic payload. Methods: Following surgical resection, freshly collected, matched tumor adjacent normal tissues were utilized to isolate fibroblasts. Exosomes were purified from the isolated fibroblasts in presence of exosome-depleted FBS media. Physico-chemical characterization (DLS, TEM), presence of exosomal marker (CD63; immunoblotting), protein concentration (Bradford assay), cellular internalization (confocal microscopy) and miR-145 expression (qRT-PCR) in the exosomes were performed. Efficacy of exosomes to deliver therapeutics was investigated utilizing our recently identified drug, ormeloxifene (ORM). Drug encapsulation and loading efficiency was performed using UPLC. Functional assays, such as, proliferation were performed using clinically relevant PanCa cell lines (HPAF-II/AsPC-1). Results: NAT derived exosomes show effective size and zeta potential (size: 44.12 ± 0.89; Zeta potential: -14.9 mV), which is ideal for drug delivery purposes. The purification of exosomes was confirmed by analyzing the expression of exosomal markers, such as CD63. Immunofluorescence for CD63 expression confirmed the efficient delivery of exosomes in PanCa cells. Our results indicated high drug loading capacity of NAT derived exosomes as demonstrated though UPLC. ORM loaded exosome treatment efficiently delivered ORM into the cancer cells and inhibited tumorigenic features such as, proliferation of PanCa cells. Additionally, NAT derived exosomes showed enhanced expression of tumor suppressor microRNA, miR-145, suggestive of their therapeutic importance. We observed restoration of lost miR-145 levels in PanCa cells on incubation with NAT derived exosomes. This further indicates relevance for their utilization in the development of novel therapeutic modalities. Conclusion: Our observations offer importance of the utilization of NAT derived exosomes for personalized medicine as a therapeutic strategy in PanCa treatment. Citation Format: Saini Setua, Sheema Khan, Murali Yallapu, Sonam Kumari, Meena Jaggi, Subhash C. Chauhan. Therapeutic intervention for pancreatic cancer using autologous exosomes [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 2110.

Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics. Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database. Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented. Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis. Citation Format: Manish K. Tripathi, Chidi Zacheaus, Kyle Doxtater, Zachary Stiles, Fatemeh Keramatnia, Nadeem Zafar, Mahul Amin, Meena Jaggi, Subhash Chauhan. MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer [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 5178.

Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortality in the United States due to very limited therapeutic options. Thus, there is an urgent need to develop novel therapeutic strategies for the management of this disease. Paclitaxel (PTX), a natural product derived from the Pacific Yew tree, is an agent typically used in combination therapy for PanCa. Currently, PTX nanoparticles (Abraxane®) along with gemcitabine are in use for the treatment of advanced PanCa. Herein, we have generated and characterized a novel nano-formulation of PTX comprised of PLGA core, stabilized with polyvinyl alcohol (PVA) and coated with poly-L-lysine (PLL). Dynamic light scattering (DLS) characterization indicated that the average particle size of this formulation was approximately 160 nm, and exhibited Zeta potentials of ~ -6.02 mV in 10% serum, along with an outstanding PL loading efficiency. We also evaluated the uptake mechanism of PPNPs in PanCa cells using different endocytosis inhibitors (Genistein, amiloride, nocodazole, chlorpromazine, and methyl beta cyclodextrin). Our results demonstrated that PPNPs were taken up mainly by lipid raft mediated endocytosis pathway in an energy-dependent manner. Next, we evaluated its therapeutic efficacy and underlying molecular mechanisms in human PanCa cells (HPAF-II, Panc1, and MiaPaCa-2) by performing various in vitro experiments. We first performed MTS and colony formation assays to determine the effects of free PTX and PPNPs on growth of PanCa cells. In this experiment, cells were treated with various concentrations of free PTX or abraxane, or PPNPs (2.5-160 nM) for 48 hrs. As a result, the PPNPs formulation showed more therapeutic effect over free abraxane as determined by decrease in cell proliferation and enhanced apoptosis induction in PanCa cells. PPNPs also showed a significant (P Citation Format: Bilal B. Hafeez, Andrew E. Massey, Vivek K. Kashyap, Mohammad Sikander, Advit Shetty, Mehdi Chaib, Hassan Mandil, Mohan Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel nano-formulation of paclitaxel for pancreatic cancer therapy [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 LB-011.

Objective: Cervical cancer is one of the most common and deadly cancers among women worldwide and is associated with persistent Human Papillomavirus (HPV) infection. Human papilloma virus (HPV) expressing E6 and E7 oncoproteins involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. Therefore, non-toxic agents that have potential to inhibit the expression of E6 and E7 oncoproteins and their regulated oncogenic signaling pathways could be used in the management of cervical cancer. Microtubule targeting agents including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers but most of these agents have toxic side effects and develop chemoresistance. Herein, we investigated the potential anti-cancer effects of a novel tubulin targeting agent (ABI-231) in in vitro and in vivo model systems. Method: ABI-231 ((2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl))- methanone was synthesized in our home institution. Human cervical cancer cells (CaSki and SiHa) were used as in vitro model system. To determine the effect of ABI-231 on cell proliferation, migration and invasion, we performed MTS and wound healing assays. Effect of ABI-231 on the expression of HPV E6/E7 was determined by Western blot, qRT-PCR, and confocal microscopy. Xenograft study was performed to determine the effect of ABI-231 on cervical tumor growth. Results: ABI-231 treatment significantly (P Conclusions: Taken together, our results demonstrate the potential anti-cancer efficacy of ABI-231 in in vivo and in vivo. ABI-231 can be explored as a potent therapeutic agent for the treatment of cervical cancer. Citation Format: Vivek K. Kashyap, Bilal B. Hafeez, Qinghai Wang, Neeraj Chauhan, Prashanth K. Nagesh, Nirnoy Dan, sonam kumari, Shabnam Malik, Saini Setua, Aditya Ganju, Murali M. Yallapu, Duane D. Miller, Wei Li, Meena Jaggi, Subhash C. Chauhan C. Chauhan. ABI-231: A novel small molecule suppresses tumor growth and metastatic phenotypes of cervical cancer cells via targeting HPV E6 and E7 [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 679.

Background: Docetaxel (DTX) is an FDA approved drug for the treatment of advanced prostate cancer (PrCa). However, chemo-resistance and toxicity limits its use in clinic. Accumulating evidence suggests that activation of cancer stem cells (CSCs) is one of the mechanisms leading to developing chemo-resistance Therefore, identification of new pharmacophores that can target CSCs may improve the therapeutic efficacy of docetaxel for the treatment of advanced PrCa. Cucurbitacin D is one of the potent analogues of cucurbitacin and has shown several health benefits including anti-cancer activity. Herein, we report that Cucurbitacin D augments the therapeutic efficacy of DTX via modulating CSCs and miR-145. Methods: Prostate cancer stem cells (pCSCs) were used as a model system to investigate the effect of Cucurbitacin D alone or in combination with docetaxel. Effect of Cucurbitacin D was evaluated on cell growth by MTS and xCELLigence assays and apoptosis induction was evaluated by flow cytometry. Colony formation assay was performed to investigate the effect of Cucurbitacin D on clonogenic potential of pCSCs cells. Effect of Cucurbitacin D on key oncogenic molecules was analyzed by Western blot and qRT-PCR analyses. To investigate the therapeutic and chemo-sensitization effect of Cucurbitacin D, we performed xenograft study using pCSCs. Results: Our results demonstrated that Cucurbitacin D potentiates the therapeutic effect of docetaxel in pCSCs. Cucurbitacin D treatment significantly inhibited growth and clonogenic potential. Moreover, Cucurbitacin D effectively sensitized the effect of docetaxel as analyzed by significant (P Conclusion: Our findings suggest that cucurbitacin D is a novel agent and has great promise to improve the therapeutic efficacy of docetaxel. Thus, cucurbitacin could be a novel therapeutic modality for the treatment of advanced prostate cancer. Citation Format: Mohammed Sikander, Shabnam Malik, Bilal Bin Hafeez, Hassan Mandil, Fathi T. Halaweish, Meena Jaggi, Subhash C. Chauhan. Cucurbitacin D enhances the therapeutic efficacy of docetaxel via targeting cancer stem cells and miR-145 [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 2934.

Objectives: Docetaxel (Dtx) is the primary choice for the treatment of castrate-resistant prostate cancer (PrCa). Besides its therapeutic benefits, many men with castrate-resistant PCa fail to respond to treatment due to Dtx resistance. There are multiple signaling mechanisms that play pivotal role in the development of Dtx resistance. Seminal reports stated that Tau, highly soluble microtubule-associated protein play a crucial role in Dtx resistance development. Disruption Tau signaling affects its chemoresistance. To revert the drug-resistance and target specific pathway, development of novel and strategic therapies are much needed. In this line, we employed magnetic nanoparticle (MNP) loaded Dtx (MNP-Dtx) to revert the chemo resistance of Dtx in PrCa. Methods: Docetaxel loaded magnetic nanoparticle (MNP-Dtx) formulation is composed of an iron oxide core coated with cyclodextrin (for drug loading) and F127 polymer (for particle stability and chemosensitization). Dtx-resistant PrCa cell line models (PC-3R and DU145R) were developed in our laboratory by repeatedly treating cancer cells in a dose escalation manner for over 24 months. Intracellular drug levels of MNP-Dtx in wild type and drug resistant cancer cells was determined by flow cytometry, TEM and HPLC methods. The superior in vitro anti-cancer activity of MNP-Dtx was examined by MTS, proliferation kinetics, colony formation, immunoblotting and qPCR studies in both wild type and drug resistant PrCa cell lines. The interaction between tau and tubulin studies were assessed through immunoprecipitation studies. Results: MNP-Dtx formulation showed optimal particle size and zeta potential which can efficiently be internalized in PrCa/Dtx resistant PrCa cells. MNP-Dtx exhibited enhanced cellular uptake in drug resistant cell lines over wild type cells in a dose and time dependent manner, with 2 to 3 folds increase. Immunoblot and q PCR studies showed the expression of Tau, whole β tubulin, β tubulin III, β tubulin IV and MDR1 were found to be deregulated in both wild type and resistant cells. To delineate the mechanism of chemosensitization by MNP-Dtx on resistant cells, we performed immunoprecipitation studies, found the expression of Tau was significantly reduced during MNP-Dtx treatments w.r.t control and Dtx. HPLC studies have shown higher amounts of Dtx levels were detected in cells after MNP-Dtx treatments, annotating the sustain drug release ability of MNP nanoformulation. Further, these results were confirmed through tubulin polymerization studies. Conclusion: Overall, sustained drug release of Dtx aided in high intracellular levels of drug, thus facilitating high probability of Dtx-tubulin interaction instead of Tau-tubulin interactions resulting in tubulin fibers stabilization. This promotes Dtx induced apoptosis in wild type and Dtx resistant PrCa cells. This novel therapeutic modality might be effective to treat patients with Dtx resistant PrCa. Citation Format: Prashanth K. B. Nagesh, Pallabita Chowdhury, Elham Hatami, Vivek K. Kashyap, Bilal B. Hafeez, Sheema Khan, Subhash C. Chauhan, Meena Jaggi, Murali M. Yallapu. Docetaxel nanoformulation reverts drug resistance in prostate cancer [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 4657.

MUC13, a transmembrane mucin, is normally expressed in gastrointestinal and airway epithelium. Its aberrant expression has been correlated with gastric colon and cancer. However, the expression and functions of MUC13 in ovarian cancer are unknown. In the present study, the expression profile and functions of MUC13 were analyzed to elucidate its potential role in ovarian cancer diagnosis and pathogenesis. A recently generated monoclonal antibody (clone PPZ0020) was used to determine the expression profile of MUC13 by immunohistochemistry using ovarian cancer tissue microarrays and 56 additional epithelial ovarian cancer (EOC) samples. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with the normal ovary/benign tissues. Among all ovarian cancer types, MUC13 expression was specifically present in EOC. For the functional analyses, a full-length MUC13 gene cloned in pcDNA3.1 was expressed in a MUC13 null ovarian cancer cell line, SKOV-3. Here, we show that the exogenous MUC13 expression induced morphologic changes, including scattering of cells. These changes were abrogated through c-Jun NH2 kinase (JNK) chemical inhibitor (SP600125) or JNK2 siRNA. Additionally, a marked reduction in cell-cell adhesion and significant (P < 0.05) increases in cell motility, proliferation, and tumorigenesis in a xenograft mouse model system were observed upon exogenous MUC13 expression. These cellular characteristics were correlated with up-regulation of HER2, p21-activated kinase 1, and p38 protein expression. Our findings show the aberrant expression of MUC13 in ovarian cancer and that its expression alters the cellular characteristics of SKOV-3 cells. This implies a significant role of MUC13 in ovarian cancer. [Cancer Res 2009;69(3):765–74]

Single antigen-targeted intraperitoneal radioimmunotherapy for ovarian cancer has shown limited success. Due to the heterogeneous expression of tumor antigens on cancer cells, a multi-antigen targeting approach appears logical to augment the therapeutic efficacy of antibody-guided therapy. In the interest of developing this novel approach, ovarian cancer tissue microarray slides containing cancer and benign/non-neoplastic tissue samples ( n = 92) were processed for single-, double-, and triple-antigen labeling using antibodies for the tumor-associated antigens TAG-72, MUC1, and CA125. Among all ovarian cancer types, 72%, 61%, and 50% of the samples showed immunolabeling for TAG-72, MUC1, and CA125, respectively. Expression level of these antigens was significantly ( p70%), a heterogeneous labeling pattern was observed (only 30–40% of the cancer cells within the sample were labeled). However, upon combining the three antigens (triple-antigen labeling), 98% of the epithelial ovarian cancer samples were labeled and >95% of the cancer cells within each sample were labeled. Our data indicate that the heterogeneous expression of cancer antigens appears to be a major obstacle in antibody-guided therapy, and this can be overcome by multiple antigen targeting. Therapeutic efficacy of antibody-guided therapy for ovarian cancer treatment will be enhanced by the combined targeting of TAG-72, MUC1, and CA125. (J Histochem Cytochem 55: 867–875, 2007)

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility and invasion of PDAC cells - all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13-HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13-HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Clinically, pancreatic tumors have proven to develop resistance to available treatment options; however, efficacy might be achieved if the required levels of drugs reach the tumor site. Tumor microenvironment (TME) leads to extensive desmoplasia and chemo-resistance, contributing immensely to the invasive and metastatic process of pancreatic carcinoma [1]. Desmoplasia involves the excessive production of extracellular matrix and is associated with proliferation of stromal cells, including myofibroblasts and stellate cells. Recent studies suggest that the Sonic hedgehog (SHH) pathway plays a key role in desmoplasia [2] and progression of cancer, including pancreatic ductal adenocarcinoma (PDAC) [3, 4]. The implementation of stromal targeting strategies in clinical practice still poses significant challenges due to vast heterogeneity in sensitivity across different cancers and even tumor types. This may be likely due to inherent differences in expression of pro-angiogenic, invasiveness factors, growth factors/receptors and differential dependency of tumors on hypoxia and nutrition. Unlike other tumor types, pancreatic tumors that are surrounded by abundant stroma have hypo-vascularity and poor perfusion that render them less dependent on vascular supply, leading to tumor growth and hindrance in drug delivery [5]. However, so far, preclinical/clinical data reveal that despite most of the hedgehog pathway inhibitors could deplete the pancreatic tumor stroma, the outcomes outweighed over the therapeutic benefit. One plausible explanation could be the result of the side effects other than the diminution of stroma. Therefore, the identification of novel modalities that could target pancreatic stroma and overcome inaccessibility of drugs is desired. Our study, published in Cancer Research [6], provides a preclinical proof of concept suggesting that ormeloxifene, a non-steroidal, might improve therapeutic outcomes in pancreatic cancer by targeting the stromal component. This study reinforces an insight that pancreatic tumors are responsive to stroma depletion, which might enhance the delivery of drugs to tumors. This concept was investigated in a pancreatic cancer cell line and xenograft mouse models. Ormeloxifene is a non-hormonal, non-steroidal molecule that has potent anti-cancer properties against various cancers such as breast cancer, head and neck cancer, and chronic myeloid leukemia [7]. The study demonstrates the combinatorial effects of ormeloxifene with gemcitabine at the molecular level and suggests that ormeloxifene targets the SHH signaling pathway and TME, thereby inhibiting proliferation and inducing death in PDAC cells. We observed that the effects displayed by ormeloxifene were more prominent and comparable to a known SHH inhibitor, GDC-0449 (SMO antagonist), in PDAC cells. The molecular alterations involved in the process included downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NFκB-65, p-AKT and Cyclin D1. Preclinical studies revealed the ability of ormeloxifene to potentiate the anti-tumorigenic effect of gemcitabine in PDAC xenograft mice. Ormeloxifene disrupted the stroma of fibrotic pancreatic tumors and inhibited the proliferating stellate and myeloid cells that are involved in the development of fibrosis. Combination treatment with gemcitabine decreased the size of tumors and significantly inhibited metastasis. Mice treated with gemcitabine had abundant stroma, which decreased when the treatment was combined with ormeloxifene. The rich stromal component was observed in control and gemcitabine-treated mice tumor tissues, while ormeloxifene alone or in combination with gemcitabine displayed markedly less stromal component and invaded stromal tissue. This was indicated by reduced numbers of stroma myofibroblasts infiltrating the tumor tissue, as indicated by reduced PSCs, αSMA, FSP, cygb/STAP, and collagen 1 expression in the tissues. Thus, this study suggests that ormeloxifene acts by remodeling the pancreatic TME by targeting the cellular populations in the stroma that may affect tumor immune-surveillance. Considering the huge fibrotic nature of pancreatic tumors and to overcome the vast heterogeneity observed within the tumors, we have prepared a PLGA [poly(lactic-co-glycolic acid)] based nanoparticle formulation of ormeloxifene and discussed its enhanced efficacy in an article published in Biomaterials [8]. The formulation efficiently escapes lysosomal degradation and demonstrates remarkable anti-cancer potential in pancreatic cancer cells and xenograft mice. The formulation remarkably potentiated the efficacy of ormeloxifene treatment by extending median survival of the mice. Our data suggest that the formulation is highly efficient for the inhibition of pancreatic tumor growth and thus can be valuable for the treatment of pancreatic cancer. Pancreatic tumors being independent of their vascular beds and viable in hypoxic, nutrition-deprived conditions require tumor specific targeted systemic delivery of administered drugs in the absence of abundant vessels. To achieve drug specificity that can render larger accumulation in the tumor area, the formulation can be conjugated with tumor specific antibodies/aptamers (for active targeting) to preferentially reach and accumulate in tumors by both passive (Enhanced Permeability and Retention (EPR) effect) and active targeting approaches (Figure ​(Figure11). Figure 1 Schematic representation of the reciprocal interactions between stromal components and pancreatic ductal adenocarcinoma In conclusion, management of TME using this novel therapeutic modality and potential delivery system might add clinical benefit to existing standard treatments utilizing combination treatment regimens. Our studies support the understanding that manipulation of TME may overcome the stromal barrier for effective treatments in pancreatic cancer.

BACKGROUND Mucins are considered important markers for early diagnosis and targeted therapy due to their aberrant and unique expression pattern during malignant progression of carcinomas. Recent findings have provided substantial evidence for the involvement of transmembrane mucins, MUC1 and MUC4, in altered cell signaling, tumor growth, and metastasis. METHODS Immunohistochemical analyses were performed on prostate tumor tissues for expression profiling of the two transmembrane mucins, MUC1 and MUC4. In cancer cell lines, the expression was studied by RT-PCR and immunoblot analyses. Cells were treated with DNA-methylase and histone-deacetylase inhibitors to examine the implication of epigenetic mechanism(s) in MUC4 regulation. RESULTS The expression of MUC4 was significantly downregulated in prostate cancer tissues (n = 38, P = 0.0026) compared to normal/benign prostatic hyperplastic regions. A faint to moderate staining was observed in 26.3% cases of cancer, while 84.2% cases of adjacent normal were positive for MUC4 with moderate to strong staining in most cases. Similar observations were made in immortalized normal prostate epithelial and cancer cell lines. MUC1 also showed a reduced expression in prostate tumor tissues; however, its expression was comparable in all normal prostate epithelial and cancer cell lines. Interestingly, we also found that epigenetic mechanism(s) might be implicated in MUC4 gene silencing. CONCLUSIONS Our data suggest that MUC4 downregulation may be of significance for diagnostic applications in prostate cancer. © 2005 Wiley-Liss, Inc.

Pancreatic cancer is the fourth most prevalent cancer with about an 85% mortality rate; thus, an utmost need exists to discover new therapeutic modalities that would enhance therapy outcomes of this disease with minimal or no side effects. Ormeloxifene (ORM), a synthetic molecule, has exhibited potent anti-cancer effects through inhibition of important oncogenic and proliferation signaling pathways. However, the anti-cancer efficacy of ORM can be further improved by developing its nanoformulation, which will also offer tumor specific targeted delivery. Therefore, we have developed a novel ORM encapsulated poly(lactic-co-glycolic acid) nanoparticle (NP) formulation (PLGA-ORM NP). This formulation was characterized for particle size, chemical composition, and drug loading efficiency, using various physico-chemical methods (TEM, FT-IR, DSC, TGA, and HPLC). Because of its facile composition, this novel formulation is compatible with antibody/aptamer conjugation to achieve tumor specific targeting. The particle size analysis of this PLGA-ORM formulation (∼100 nm) indicates that this formulation can preferentially reach and accumulate in tumors by the Enhanced Permeability and Retention (EPR) effect. Cellular uptake and internalization studies demonstrate that PLGA-ORM NPs escape lysosomal degradation, providing efficient endosomal release to cytosol. PLGA-ORM NPs showed remarkable anti-cancer potential in various pancreatic cancer cells (HPAF-II, AsPC-1, BxPC-3, Panc-1, and MiaPaca) and a BxPC-3 xenograft mice model resulting in increased animal survival. PLGA-ORM NPs suppressed pancreatic tumor growth via suppression of Akt phosphorylation and expression of MUC1, HER2, PCNA, CK19 and CD31. This study suggests that the PLGA-ORM formulation is highly efficient for the inhibition of pancreatic tumor growth and thus can be valuable for the treatment of pancreatic cancer in the future.