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A breast biopsy marker is a very small object that is introduced into the breast to serve as a tissue marker. The placement of a breast marker following a biopsy or to mark an abnormality in the breast has become standard practice in the clinical setting. Breast biopsy markers offer a wide range of benefits which includes the prevention of re-biopsy of a benign tumor, differentiating multiple lesions within the breast, evaluation of the extent of a tumor, and increased precision during surgery.This review article presents a range of breast biopsy markers used in clinical practice. First, an overview of the necessity of breast markers in healthy breast management. Second, it summarizes the diversity in composition, shape, unique properties and features, and bio-absorbable carriers of breast biopsy markers. Finally, it also discusses the possible use of clinically approved breast biopsy markers in various scenarios and their implications.This review serves as a guide in the selection of an appropriate breast marker. We believe that some of the common drawbacks associated with current breast biopsy markers can be overcome by developing novel polymer-metal and composite-based breast biopsy markers.

Effect of ORM on cell cycle analysis and β-catenin localization. A. Histogram (i) and table (ii) represent the cell cycle distribution in DU145 cells. B. Confocal images of β-catenin, DAPI and merged images of control (i) and ORM treated (ii) PC3 cells.

PDF file - 126K, MNP-CUR nanoparticles efficiently internalize in cancer cells.

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Background: A global review on oral health by the World Health Organization (WHO) emphasized that despite great improvements in the oral health of populations in several countries, global problems still persist. Dental caries and periodontal disease have historically been considered the most important global oral health burdens. This is particularly so among underprivileged groups in both developing and developed countries. The application of natural products for the control of oral diseases is considered as an interesting alternative to synthetic antimicrobials due to their lower negative impact, and for the effort to overcome primary or secondary resistance to the drug during therapy. Objective: To review the current evidence on the antimicrobial efficacy of plant extracts on dental caries and plaque microorganisms. Materials and Methods: A literature search was made for 6 months in PubMed, PubMed Central, MEDLINE, LILACS/BBO, Cochrane database of systematic reviews, SCIENCE DIRECT, and Google scholar databases. The results from the relevant published literatures are discussed. Summary and Conclusion: The extracts of Azadirachta Indica, Ocimum sanctum, Murraya koenigii L., Acacia nilotica, Eucalyptus camaldulensis, Hibiscus sabdariffa, Mangifera indica, Psidium guajava, Rosa indica, and Aloe barbadensis The current evidence is on individual plant extracts against bacteria involved in either caries or periodontitis. The research assessing the antimicrobial efficacy of a combination of these plant extracts against dental caries and periodontal pathogens is the need of the hour, and such research will aid in the development of a novel, innovative method that can simultaneously inhibit two of the most common dental diseases of mankind, besides slowing the development of drug resistance.

Background: Epidemiological observations such as mental retardation, physical deformities, etc., in children besides different types of cancer in the adult population of the Malwa region have been reported. The present study is designed to get insight into the role of naturally occurring radioactive material (NORM) in causing detrimental health effects observed in the general population of this region.Materials and Methods: Deep soil samples were collected from different locations in the Malwa region. Their activity concentrations were determined using low-level background gammaray spectrometry. High efficiency and high purity germanium detector capped in a lead-shielded chamber having a resolution of 1.8 keV at 1,173 keV and 2.0 keV at the 1,332 keV line of 60Co was used in the present work. Data were evaluated with Genie-2000 software.Results and Discussion: Mean activity concentrations of 238U, 232Th, and 40K in deep soil were found to be 101.3 Bq/kg, 65.8 Bq/kg, and 688.6 Bq/kg, respectively. The mean activity concentration of 238U was found to be three and half times higher than the global average prescribed by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). It was further observed that the activity concentration of 232Th and 40K has a magnitude that is nearly one and half times higher than the global average prescribed by UNSCEAR. In addition, the radioisotope 137Cs which is likely to have its origin in radiation fallout was also observed. It is postulated that the NORM present in high quantity in deep soil somehow get mobilized into the water aquifers used by the general population and thereby causing harmful health problems.Conclusion: It can be stated that the present work has been able to demonstrate the use of low background gamma-ray spectrometry to understand the role of NORM in causing health-related effects in a general population of the Malwa region of Punjab, India.

Supplementary Figure S4. Schematic representation of the treatment regime of xenograft mice.

The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer. Cancer Res; 75(11); 2292–304. ©2015 AACR.

Legends for Supplementary Figures S1-S4.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective Candida auris is a multidrug-resistant pathogen that presents a serious global threat to human health. The U.S. Centers for Disease Control and Prevention has classified C. auris as an urgent threat to public health due to its clinical and economic impact and future projections of new infections over the next 10 years. Candida auris infections are difficult to treat since many isolates display high levels of resistance to fluconazole and exhibit variable resistance to amphotericin B and echinocandins. In this study, we performed comparative transcriptomics to understand the molecular mechanisms associated with azole-resistance in C. auris environmental isolates. Material and Methods Two sets of environmental isolates including azole-resistant (n = 2) and azole susceptible (n = 1) isolates were used for RNA-Seq analysis. Pair-wise comparisons in edgeR were used for comparing the number of differentially expressed genes (DEGs) between the azole susceptible and resistant isolates. GO term enrichment analysis was performed using the ‘enrichGO’ function from the cluster Profiler package. Only GO categories with a q-value Results Our data show significant enrichment of ergosterol biosynthesis genes, drug transport, MAPK pathway as well as chromatin remodeling genes in azole-resistant strains compared to susceptible isolates. A total of 468 and 564 differentially expressed genes were identified in two azole-resistant isolates compared with the susceptible strain. A large number of multidrug transporter genes (CDR1, MDR1, HGT2, HGT7, HGT13, HGT17, and NGT1) were differentially expressed between the two sets of strains. Interestingly, the overexpression of ERG11 (azole target gene), and CDR1 (drug transporter) genes was observed in resistant isolates as compared with susceptible strain. Furthermore, resistant strain has two copies of ERG11 while susceptible isolate has single copy of ERG11. Notably, 8/21 genes involved in the ergosterol biosynthesis pathway were found to be induced in azole resistant isolates. These include HMG1, ERG1, ERG2, ERG3, ERG6, ERG10, ERG13, and ERG25. Furthermore, other multidrug transporters MDR1 and SNQ2 responsible for azole resistance in other Candida species like C. glabrata also showed significant expression changes between the two sets of isolates. Furthermore, HGT7 (glucose transporter) and NGT1, (N-acetyl glucosamine transporter) genes associated with azole and polyene resistance were found to be upregulated in the resistant isolate as compared with susceptible strain. Additionally, a Glycophosphatidylinositol (GPI)-anchored protein unique for C. auris, PGA7 was found to be overexpressed in resistant isolate. Importantly, we also identified several secreted aspartic proteases (SAP3, SAP5, SAP8, and SAP9) to be downregulated between the two sets. Conclusion The present study identifies several gene families that are differentially expressed in azole resistant vs susceptible C. auris strains. These findings suggest that azole-resistance in C. auris environmental isolates is influenced by changes in cell wall, lipid, and ergosterol biosynthesis. Overall, these data provide a framework for the mechanistic understanding of azole resistance mechanisms in C. auris environmental isolates.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Candida krusei accounts for 2.8% of invasive candidiasis worldwide. Fluconazole resistance and its underlying mechanism in clinical isolates of C. krusei (n = 137) collected from eight hospitals in India were investigated. Also, genetic diversity of C. krusei strains among different hospitals was studied through short tandem repeat (STR) genotyping. Material and Method All the isolates were identified by MALDI-TOF MS. Antifungal susceptibility test was done by using broth microdilution method (CLSI-M27). To evaluate the genetic relatedness among the strains, STR typing was done by using 9 STR markers. To understand the fluconazole-resistant mechanisms in C. krusei, known fluconazole resistance mechanisms such as alterations in target enzyme ERG11 and drug transporters ABC1, and ABC2 were investigated in 35 C. krusei isolates [18 fluconazole-susceptible (FLU-S), and 17 fluconazole-susceptible dose-dependent (FLU-SDD)]. Furthermore, transcriptomics of one FLU-SDD (MIC 32 mg/L) and one FLU-S (MIC 4 mg/l) isolate was performed. Results Majority (77%) of C. krusei isolates were from bloodstream infections. Notably, 70% of candidemia cases occurred in neonatal intensive care units (NICUs). Remarkably, 81% (n = 110) were detected as fluconazole-SDD (MIC 16-32 mg/l), and the remaining 19% were FLU-S (MIC ≤ 8 mg/l). Marked genetic diversity with 51 diverse STR types was noticed among the 106 isolates. Interestingly, two ongoing candidemia outbreaks were observed in two geographically separated hospitals both representing NICU isolates. In addition, a large cluster containing isolates from six different hospitals was observed. ERG11 mutation analysis revealed that it did not harbor any mutation contributing to the flu-resistance. Overexpression of the ABC1 gene in 11 FLU-SDD isolates out of 17 as compared to FLU-S isolates was noted. However, no alteration was observed in the expression of ERG11 and ABC2 in both groups. Transcriptomics analysis revealed a significant number of differentially regulated genes were distributed in various gene-ontology terms including transport (10 genes), mitogen-activated protein kinase (MAPK) signaling (8 genes, MSG5, PTP3, STE50, BNR1, OPY2, STE5, SKN7, and RLM1), ergosterol biosynthesis (3 genes, ERG24, ERG25, and ERG26) and transcription factors (7 genes). In addition to the up-regulation of ergosterol pathway genes, overexpression of key transcriptional regulator of ergosterol biosynthesis genes UPC2 was observed in FLU-SDD isolates as compared with susceptible. Additionally, FLU-SDD isolate showed 2-fold increased expression of PDR12, plasma membrane ATP-binding cassette (ABC) transporter. Next, ICL1 (Isocitrate Lyase), a major glyoxylate-synthesizing enzyme was found to be 5-fold down-regulated in FLU SDD isolate compared to susceptible. The loss of ICL1 alters the expression of the FKS1, ERG11, and CDR2 genes in C. albicans. Taken together, the increased expression of PDR12 and altered MAPK singling network may partially account for the FLU resistance in C. krusei FLU-SDD isolate. Conclusion Candida krusei isolates among different hospitals showed large genetic diversity (54 different genotypes). Also, the presence of C. krusei clonal strains in six different hospitals suggests possible introduction from a widespread environmental source and human-to-human transmission. In comparison to other Candida species, the resistant mechanism in C. krusei seems to be more complex. Therefore, an in-depth study of other resistance mechanism pathways in C. krusei is further warranted.

Candida albicans is a normal component of the human microflora that colonizes mucosal/epithelial surfaces and the gastrointestinal tract as a commensal organism. However, in an immunocompromised host, it can cause life-threatening infections of high mortality and morbidity. Virulence as well as antifungal drug resistance of C. albicans is often regulated by posttranslational modifications (PTM) of proteins via lysine acetylation by lysine acetyltransferases. Here, we report an experimental approach using tandem mass tag (TMT) labeling for the detection and quantification of lysine acetylation of histone and nonhistone proteins in C. albicans.

Pancreatic cancer (PanCa) is characterized by lack of early diagnosis, poor response to available therapeutic modalities and chemoresistance. Gemcitabine (GEM) is currently considered most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. This poor drug response has been attributed to desmoplasia, causes suboptimal drug delivery, alters tumor microenvironment (TME), which includes tumor surrounding blood vessels, fibroblasts, immune cells, extracellular matrix, and other signaling molecules and induces chemo-resistance in tumors. To overcome these existing issues associated with chemotherapy, identification and development of novel therapeutic modalities are a pressing need. Piperlongumine (PL) is a natural alkaloid isolated from the long pepper, Piper longum L., and has shown substantial cancer-preventive and therapeutic efficacy against a variety of cancers. However, delivering its effective concentration in pancreatic tumors has been challenging. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated piperlongumine loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PLGA-PL), which effectively inhibits the growth of PanCa cells. In this study, we demonstrate that PLGA-PL effectively sensitize tumor cells to GEM via decreased desmoplasia, altered TME, SHH/CXCL12/CXCR4 and immune surveillance. Our finding show that PLGA-PL synergizes with GEM in inhibiting PanCa cell (HPAF-II and Panc-1) growth, migration, and invasion compared to free PL. Mechanistically, PLGA-PL targets the TME via inhibition of sonic hedgehog (SHH) pathway and oncogenic CXCR4/CXCL12 signaling axis that inhibits bidirectional tumor-stromal cells interaction. We have also found that PLGA-PL alone and in combination with GEM targets cancer stem cells by inhibiting pluripotency maintaining stemness factors (Nanog, Sox2, c-Myc, CD133, and Oct-4) as determined by qRT-PCR, Western blotting, and immunofluorescence analysis, and further confirmed by restricting tumor sphere formation. Furthermore, PLGA-PL also effectively targets tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in presence of PLGA-PL or vehicle control. In additions, PLGA-PL effectively increases phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that PLGA-PL effectively targets TME, facilitates GEM uptake by inhibiting the activation of CXCR4/CXCL12/SHH signaling, and reprograming the tumor immune surveillance. This study suggests that PLGA-PL has great potential for future clinical use in management of PanCa. Citation Format: Vivek Kumar Kashyap, Neeraj Chauhan, Mohammed Sikander, FNU Pranav, Rahul Tiwari, Bilal B. Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Piperlongumine nanoformulation enhances gemcitabine therapeutic response in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 823.

Background: Adequate bioimaging is crucial in cancer management in many ways including screening, detection, characterization, staging and grading, therapy response, surgical guidance, and margins assessment. Indocyanine green (ICG) is one of the FDA-approved near infra-red fluorescent (NIRF) probe for cancer imaging and image-guided surgery in clinical setting. However, limitations of ICG includes poor photostability, high concentration toxicity, short circulation time, and poor cancer cell specificity. To overcome these hurdles, we engineered a nanoconstruct composed of poly(vinyl pyrrolidone) (PVP)-indocyanine green that is cloaked self-assembled with tannic acid (termed as ICG-Glow NPs) for the cancer cells/tissues specific targeting. Methods: Pursuing the novel nanotherapy approach, our lab has developed PVP-TA based ICG (PVT-ICG) fluorescent nanoparticles via self-assembly process. Our optimized PVT-ICG nanoformulation was further characterized for its physicochemical properties. An IVIS imaging system was further used to measure NIR fluorescence of novel PVT-ICG. Moreover, Human cancer (Breast, Pancreatic, Liver and Prostate) tissue microarrays (TMAs) were histochemically stained to assess cancer cell targeting/specificity of PVT-ICG. Results: PVT-ICG indicated particle size and surface charge ideal for cancer cell/tissue delivery. PVT-ICG, further, demonstrated improved photostability and fluorescent intensity. Additionally, TMA studies exhibited enhanced internalization and cancer targeting/specificity of PVT-ICG nanoparticles compared to free ICG dye in all cancers. Conclusion: Collectively, our findings suggest that this NIR fluorescent probe PVT-ICG has great potential for becoming a novel and safe imaging modality for various types of cancer cells and tumors which can result in early cancer diagnosis leading to improved disease management. Citation Format: Neeraj Chauhan, Marco Cabrera, Pallabita Chowdhury, Prashanth K. Nagesh, Anupam Dhasmana, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Illuminating cancer cells with a novel nano fluorescent NIR probe for bioimaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 826.

Candida auris is an emerging multidrug-resistant human fungal pathogen often refractory to treatment by all classes of antifungal drugs. Amphotericin B (AmB) is a fungicidal drug that, despite its toxic side effects, remains a drug of choice for the treatment of drug-resistant fungal infections, including those caused by C. auris. However, the molecular mechanisms underlying AmB resistance are poorly understood. In this study, we present data that suggests membrane lipid alterations and chromatin modifications are critical processes that may contribute to or cause adaptive AmB resistance in clinical C. auris isolates. To determine the plausible cause of increased AmB resistance, we performed RNA-seq of AmB-resistant and sensitive C. auris isolates. Remarkably, AmB-resistant strains show a pronounced enrichment of genes involved in lipid and ergosterol biosynthesis, adhesion, drug transport as well as chromatin remodeling. The transcriptomics data confirm increased adhesion and reduced lipid membrane permeability of AmB-resistant strains compared to the sensitive isolates. The AmB-resistant strains also display hyper-resistance to cell wall perturbing agents, including Congo red, calcofluor white and caffeine. Additionally, we noticed an increased phosphorylation of Mkc1 cell integrity MAP kinase upon AmB treatment. Collectively, these data identify differences in the transcriptional landscapes of AmB-resistant versus AmB-sensitive isolates and provide a framework for the mechanistic understanding of AmB resistance in C. auris.

Cervical cancer (CxCa) remains the fourth leading cause of cancer related deaths among women worldwide. Cervical cancer is mainly (~ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 are the two main oncoproteins that interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Additionally, PI3K-Akt is a cell survival pathway that is aberrantly expressed in cervical cancer cells. This pathway has a profound role in inhibiting mitochondrial intrinsic apoptotic signaling pathway. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene (ORM) is a non-hormonal, anti-estrogen, oral contraceptive for human use. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in a variety of cancers. Developing nanoformulation of drugs has received much attention lately as nanoparticles have site specific targeted drug delivery. Nanoparticles have a specific size range that makes them capable of being entrapped and accumulated at the tumor site due to its leaky vasculature. As a result of it, drug is released from the particle core at a sustained rate; therefore, nanoparticulates offer enhanced bioavailability and better therapeutic efficacy. Considering these benefits, we engineered ormeloxifene loaded PLGA based novel nanoformulation (PLGA-ORM). In this work we validated anti-cancer properties of free ORM and its PLGA based nanoformulation. Our set of data showed that ormeloxifene significantly decreased the cellular proliferation and clonogenic potential of cervical cancer cells. Ormeloxifene also reduced the cellular motility and induced the apoptosis via targeting PI3K-Akt signaling in these cells. Furthermore, ormeloxifene modulated the HPV induced oncogenesis in Caski cells. Ormeloxifene also showed additive inhibitory effects on cellular proliferation and growth when used with radiation. Moreover, our novel PLGA-ORM had a particle size range of 100 – 280 nm and also exhibited excellent encapsulation of ormeloxifene in to PLGA core. PLGA-ORM was labeled with Coumarin 6 (green fluorescent) dye for its uptake studies, where PLGA-ORM internalized in cervical cancer cells in dose, time and energy dependent manner via endocytosis pathway. PLGA-ORM showed improved anti-proliferative/growth properties than free ormeloxifene in cervical cancer cells. When utilized in animals (an orthotopic mouse model) both ormeloxifene and PLGA-ORM showed great anti-tumorous properties, however PLGA-ORM had improved inhibitory effects on tumor growth than free ormeloxifene. To conclude, ormeloxifene and its nanoformulation have the potential to be a novel treatment modality for cervical cancer which can reduce the overall disease burden and improve patients’ life expectancy.

Complex Sphingolipids (SLs) are unique to fungi, which apart from being novel drug targets, also appear to act as molecular signals, in diverse biological processes. In this study, we have specifically blocked the key synthesis step of SLs metabolism by disruption of the uncharacterized CgIPT1 gene, which based on homology with other Candida spp., predicted to mediate the conversion of MIPC to M(IP)2C. We followed fusion based PCR homologous recombination method for IPT1 deletion by using dominant markerNAT1. The knockout was selected on a nourseothricin drug plate and confirmed by gene specific PCR and by checking M(IP)2C levels. We observed that the specific accumulation of MIPC or lack of M(IP)2C in C. glabrata displayed increased susceptibility to both imidazole’s (ketoconazole, miconazole and clotrimazole) and triazoles (fluconazole, itraconazole and posaconazole). RNA Sequencing of Cgipt1Δcells revealed no major impact on of expression levels of common MDR determinants albeit a distinct imbalances in expression of lipid homeostasis genes was evident. The Fluorescence Recovery after Photobleaching (FRAP) experiments confirmed that plasma membrane in Cgipt1Δ cells display a reduction in micro-viscosity leading to increase in drug diffusion and susceptibility of Cgipt1Δcells. Interestingly, the Cgipt1Δ also exhibit attenuated virulence in a murine model. Together, our data confirms the relevance of M(IP)2C in governing drug susceptibility and virulence in C. glabrata.

Alternative gene splicing (AS) is a process by which a single gene can give rise to different protein isoforms, generating proteome diversity. Despite recent advances in our understanding of AS in basic cellular processes, the role of AS in drug resistance and fungal pathogenesis is poorly understood. In Candida albicans, approximately 6% of the genes contain introns. Considering this low and random distribution of introns, we focused our study on alternative splicing (AS) and its impact on the development of drug resistance, an area largely unexplored in this yeast. We performed comparative RNA sequencing of sequential isogenic azole sensitive and resistant isolates of C. albicans. The analysis revealed differential expression of splice junctions/isoforms in 14 genes, between the drug sensitive and resistant isolates. Furthermore, C. albicans WT cells exposed to antifungal drugs, heat stress or metal deficiency also showed differential expression of isoforms for the genes undergoing AS. In this study we present data on the effect of AS on the function of SOD3. The C. albicans SOD3 has a single intron and is important for the removal of superoxide radicals. The overexpression of the two isoforms of SOD3 in its null background highlighted importance of spliced isoform in complementing the susceptibility to menadione. However, the two isoforms did not differ in rescuing the susceptibility of sod3Δ/Δto Amphotericin B. Collectively, these data suggest that AS may be a novel mechanism in C. albicans for stress adaptation and overcoming drug resistance.

Pancreatic cancer (PanCa) is the third leading cause of cancer-related deaths in the United States with limited therapeutic options available. Gemcitabine (GEM), a deoxycytidine nucleoside analog is currently considered the most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. PanCa cells are addicted to ribosome biogenesis (RiBi) which supports their high rate of growth and proliferation. Therefore, strategically targeting the process of RiBi could be one of the ideal strategies for the prevention and treatment of PanCa. In this study, we for the first-time report that RPA194, a catalytic subunit of RNA Pol I is differentially expressed in normal pancreatic ductal epithelial (NPDE) and cancer cells. We also observed differential expression of RPA194 in various grades of pancreatic tumor tissues and its level was increased in high-grade pancreatic tumor tissues. We tested our hypothesis that targeting RPA194 with a non-toxic pharmacological inhibitor (BMH-21) will inhibit the growth of advanced PanCa. We observed that BMH-21 inhibits the growth and induced apoptosis of PanCa cells in a dose-dependent manner and this inhibition was correlated with the expression of RPA194. We observed proteasomal mediated degradation of RPA194 in PanCa cells. BMH-21 treatment inhibited RPA194 occupancy on rDNA but did not affect other nucleolar proteins (UBF, fibrillarin, nucleolin, and nucleophosmin). Two times the effective concentration of BMH-21 (5 µM) did not show any cytotoxic effect in NPDE cells. However, BMH-21 degraded RPA 194 protein in these cells in a similar manner as in PanCa cells. Therefore, we speculate that other proteins are involved in the selective toxicity of BMH-21 in PanCa cells. These findings were translated to in vivo model systems. We observed that BMH-21 significantly (P Citation Format: Mudassier Ahmad, Carlos Perez, Andrew Massey, Vivek Kashyap, Neeraj Chauhan, Haider Ahsan, Jasmine Jasmine, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis addition is a novel strategy for pancreatic cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B035.

Prostate cancer (PrCa) is the most common type of cancer among men in the United States. The metastatic and advanced PrCa develops drug resistance to current regimens which accounts for the poor management. microRNAs (miRNAs) have been well-documented for their diagnostic, prognostic, and therapeutic roles in various human cancers. Recent literature confirmed that microRNA-205 (miR-205) has been established as one of the tumor suppressors in PrCa. miR-205 regulates number of cellular functions, such as proliferation, invasion, migration/metastasis, and apoptosis. It is also evident that miR-205 can serve as a key biomarker in diagnostic, prognostic, and therapy of PrCa. Therefore, in this review, we will provide an overview of tumor suppressive role of miR-205 in PrCa. This work also outlines miR-205's specific role in targeted mechanisms for chemosensitization and radiosensitization in PrCa. A facile approach of delivery paths for successful clinical translation is documented. Together, all these studies provide a novel insight of miR-205 as an adjuvant agent for reducing the widening gaps in clinical outcome of PrCa patients.

Background: Lung cancer is a predominant cause of morbidity and mortality across the world. Among all types of cancers, lung cancer is the leading cause of cancer mortalities in the United States. Non-small cell lung cancer is one of the dominant divisions of lung cancer that solely represents 85-90% of all lung cancer cases. Up to the present time, primary treatment regimens for this cancer include radiation therapy, chemotherapy, and surgery, with chemotherapy being the best option thus far. Despite the eminent benefits of chemotherapy, its value is offset by severe side effects such as renal and/or hepatic toxicity or insufficient amounts of drug available at the target site. Such pitfalls can be handled by employing natural compounds (polyphenols, phytochemicals, xanthonoid, etc.,) as chemopreventives or chemosensitizers which can improve chemotherapy activity while reducing its systemic side effects and drug resistance. To this end, our recent efforts demonstrated a synergistic therapeutic benefit of gambogic acid (GA) and gemcitabine (Gem) against lung cancer. However, simultaneous delivery of these two drugs at the tumor site is highly challenging. Therefore, development of an injectable nanoformulation that can effectively deliver both hydrophobic (GA) and hydrophilic (Gem) drugs in one formulation is a clinically unmet need. Methods: Pursuing the novel nanotherapy approach, our lab has developed an in-situ biodegradable and biocompatible human serum albumin (HSA) and tannic acid (TA) mediated complexed GA and Gem nanoparticles (G-G@HTA NPs) using the solvent evaporation method. G-G@HTA NPs formation was confirmed by particle size, FT-IR, and H-NMR. A superior therapeutic activity of G-G@HTA NPs was demonstrated by multiple in vitro functional assays as well as in an animal mouse model. Results: Our results confirmed that G-G@HTA NPs have the ideal particle size and surface charge for cancer cell/tissue delivery which can clearly be evident by preferential uptake of these nanoparticles in lung cancer cells. Further, G-G@HTA NPs superiorly inhibited cell proliferation and clonogenicity of NSCLC cells. Additionally, G-G@HTA NPs revealed an obvious and precise targeting of tumors in vivo. The promoted and more synergistic anti-tumor efficacy of G-G@HTA NPs was attained than that of combined treatments and single drugs treatments. These events were resulted with no apparent systemic and organ toxicities. Conclusion: In summary, this study details the design of a novel nanocarrier and provides an optimized strategy for constructing dual-loaded nanoparticles using a biodegradable, non-toxic, human serum albumin-tannic acid-based platform for GA and Gem co-delivery in the treatment of NSCLC which confirms a strong feasibility to implement such synergistic nanomedicine regimen in pre-clinical and clinical translations in future. Citation Format: Elham Hatami, Prashanth K. Nagesh, Neeraj Chauhan, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. A novel in-situ nanoparticle self-assembly for combination delivery of therapeutics to non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5073.

Introduction: The tumor microenvironment is a dynamic domain and communication hub for signaling molecules, contributing to proliferative and metastatic behaviors observed in cancerous tissues. Intercellular interactions are commonly mediated through extracellular vesicles that carry vital information for growth and survival, a feature that is highly advantageous for cancer cells to hijack. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing types of cancer in the United States. Often aggressive, HCC arises from the uncontrollable division of hepatocytes, which may disrupt the regular functions of the liver and other organs. Identifying biomarkers is essential to potentiate drug treatments while minimizing chances of metastasis, recurrence, and mortality. Highly proliferative cells are known to secrete exosomes containing tumor-enhancing biomolecules that contribute to cancerous progression. Thus, analyzing exosomal cargo in serum is especially attractive for early diagnostics, profiling, and therapeutic purposes. We have explored potential oncogenic lncRNAs and extracellular signaling proteins in HCC and conducted phenotypic studies to observe their mode of action. Methods: HCC cell lines were propagated in EMEM containing exosome-free FBS. Exosomes were harvested with ThermoFisher Total Exosome Isolation Reagent from cell culture media, then resuspended in PBS. Zetasizer was used to measure the sizes of harvested exosomes, and western blot analysis was used to confirm exosomal markers. UCA1 and MALAT1 were checked via RTPCR via exosomes spiked with bacterial RNA as the loading control. Bradford assays were performed on exosomes lysed with RIPA for quantifying cargo proteins. Exosomal POTE-2 was observed through western blot analysis, in addition to ELISAs to identify their presence in media. Phenotypic studies were performed using isolated exosomes. Results: The isolated exosomes were within the size range of 40-200nm, with a negative charge. The western blot of the exosomal markers confirmed their purity. RTPCR results showed LncRNAs MALAT1 and lncRNA UCA1 present in exosomes, whereas POTE-2 protein presence was confirmed with western blot. Phenotypic studies from the isolated exosomes are in progress. Conclusions: Exosomes in serum are easily accessible biomolecules making them functional, less invasive targets for profiling, diagnostics, and therapeutics via liquid biopsies. Exosome-mediated delivery of lncRNAs MALAT1 and UCA1 to primary CRC cells has been found to promote malignancy. We have verified their presence in exosomes secreted by metastatic cell lines, in addition to oncoprotein POTE-2, which hold numerous significances for our phenotypic studies. These findings reaffirm exosomes as essential contributors to disease pathogenesis and present them as promising targets for diagnostics and treatment. Citation Format: Adithya Anilkumar, Samantha Lopez, Sophia Leslie, Kyle Doxtater, Neeraj Chauhan, Bilal Hafeez, Murali Yallapu, Meena Jaggi, Subhash Chauhan, Manish Tripathi. Exosomes as nanocarriers for biomolecules and potential diagnostic targets in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2790.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States with limited therapeutic approaches. Dysregulation of the ribosome biogenesis process was observed in various cancer including pancreatic cancer. Therefore, strategically targeting ribosome biogenesis could be a novel approach for pancreatic cancer treatment. One such approach is to target ribosome biogenesis is via small molecule inhibitors of RNA Polymerase I as these inhibitors have shown promising anti-cancer activity in pre-clinical and clinical studies. We observed differential constitutive expression of various ribosomal proteins including RPA-194 in human PanCa cells when compared with normal HPDE cells. Therefore, we hypothesized that targeting RNA pol I will inhibit the growth of pancreatic cancer. In this study, we for the first time evaluated the therapeutic effect of a novel RNA polymerase I inhibitor (BMH-21) against PanCa in pre-clinical model systems. BMH-21 significantly inhibited the ribosome biogenesis process in various pancreatic cancer cells (Panc-1, AsPC1, BxPC3, MiaPaCa-2) as determined by inhibition of rRNA synthesis and POL I occupancy on rDNA. BMH-21 treatment of pancreatic cancer cells significantly degraded RPA194 protein as determined by Western blot and confocal microscopy analysis. BMH-21 treatment (0.25-5µM) showed differential IC50 in various pancreatic cancer cells which were correlated with the expression of its target proteins RPA194 and RPA135 expression. In our flow cytometry experiment, we observed a significant G0/G1 cell cycle arrest and increase Annexin V and Annexin V7-AAD positive cells in BMH-21 treated pancreatic cancer cells as compared to the vehicle treatment group suggesting its apoptosis-inducing potential in pancreatic cancer cells. BMH-21 treatment inhibited growth and metastatic phenotypes of various PanCa cells as analyzed by various functional assays (colony formation and atomic force microscopy analyses). We also evaluated the effect of BMH-21 on various kinases, which regulate ribosome biogenesis. Interestingly, we observed BMH-21 significantly (P Citation Format: Carlos Perez, Mudassier Ahmad, Andrew Massey, Asif Shahriar, Emmanuel Anning, Vivek Kashyap, Neeraj Chauhan, Anupam Dhasmana, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis is a novel strategy to suppress the growth of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3950.

Background: Pancreatic cancer remains 3rd deadliest disease, with less than 7-10% survival rate. Little progress has been seen in patient’s outcome due to high desmoplasia and chemo-resistance. Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate fibrotic tumor leading to insufficient availability of therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, that makes it an excellent protein tumor target. This study is unique as it utilizes MUC13Ab for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drug (curcumin), which would help in improving immunotherapy response. Methods: The inhouse generated MUC13Ab have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). Conjugation efficiency of the SPION-Anti-MUC13 particles was seen through cell uptake studies, by measuring fluorescence intensity, Prussian blue staining. Invasion assay and migration assay was carried out on KPC cells. We have used female C57BL/6J black mice, orthotopic mice model for investigating targeting efficacy of MUC13-SPION-CUR. Immune checkpoint therapy (PDL-1 and CTLA-4) was administrated along with MUC13-SPION-CUR and conjugated with fluorescent indocyanine green (ICG) dye for monitoring the tumor growth. Further, immunostimulatory effect of the nano formulation was done using flow cytometry. Results: Our results showed that MUC13Ab conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic syngeneic mouse model of PDAC as indicated by ICG fluorescence. Additionally, the combination formulation inhibited the tumor growth and showed more survival rate with CTLA-4. The combined treatment with CTLA-4 increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 43% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 23.8% (FoxP3+CD25+CD45+CD3+) in KrasG12D; LSL-Trp53R172H syngeneic mouse model of PDAC. Similar results were observed in SP-CUR-M13+PDL-1 group, which showed reduction in MDSCs (by 26.6%) and Tregs (by 0.1%) as compared with PDL-1 alone. Conclusion: The formulation softens up the tumors for therapies that resulted in improved response to checkpoint immunotherapies in a pancreatic orthotopic mice model. Therefore, this study indicates high significance of MUC13-SPIONS-CUR for achieving pancreatic tumor specific delivery of drugs. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients. Citation Format: Poornima Shaji, Ana Martinez, Melida Flores Cantu, Anupam Dhasmana, Neeraj Chauhan, Fnu Shabnam, Vincent Diego, Meena Jaggi, Stephen Behrman, Murali M. Yallapu, Subhash C. Chauhan, Sheema Khan. A novel approach to target tumor immune microenvironment and improve checkpoint immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB007.

Background: Liver cancer is the third leading cause of mortality attributed to cancer. Hepatocellular carcinoma (HCC) is the most prevalent form among various histological types accounting for 85% to 90% of primary liver cancers. As per Texas Cancer Registry, the Hispanic population had an incidence of 21.2 per 100,000 in Texas. The Rio Grande Valley, located in South Texas, is an underserved area composed highly of Hispanics. It faces multiple disparities that can attribute to increased risk factors of HCC and, thus, a higher liver cancer incidence and mortality. Due to this, there is a need to identify a new, inexpensive, and faster diagnostic biomarker in liver cancer. We have recently identified an extracellular secreted cancer antigen POTE-2, a member of the ankyrin domain-containing POTE gene family. Our preliminary data indicates high POTE-2 expression in HCC tumors. In this study, we will discuss the role of POTE-2 in HCC progression and its associated regulatory pathways. Methods: The Cancer Genome Atlas (TCGA) database of HCC patients (n=371 tumor; n=50 normal) was analyzed. Liver cancer cell lines procured from ATCC were analyzed for POTE-2 mRNA and protein expression through RT-PCR and western blot, respectively. Lentiviral-based plasmids were used for overexpression and knockdown studies for oncogenic assays. Localization of POTE-2, YAP1, and PDL1 was identified with immunofluorescence. Results: Comprehensive analysis of the TCGA database showed increased POTE-2 expression in tumors and is upregulated in all stages of HCC. Lentiviral transduction led to a change in phenotype in oncogenic assays. Modulation of POTE-2 expression led to changes in kinase activity and regulatory pathways. Immunofluorescence showed nuclear localization of YAP1 and PDL1 in the liver cancer cell lines. Conclusion: These studies will help discover novel mechanisms of POTE-2 protein function, signaling pathways, and its role in liver cancer progression. Citation Format: Samantha Lopez, Adithya Anilkumar, Kyle Doxtater, Sudhir Kotnala, Neeraj Chauhan, Murali Yallapu, Meena Jaggi, Subhash Chauhan, Manish Tripathi. Role of an ankyrin domain protein in hepatocellular carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 544.

Background: Pancreatic cancer (PanCa) is expected to be the second leading cause of cancer-related death by 2030. The treatment of PanCa is highly challenging due to the extremely poor response to existing therapeutic options. Highly desmoplastic tumor microenvironment in pancreatic tumor causes suboptimal delivery of therapeutic agents in tumors that eventually resulted to chemo-resistance. Piperlongumine (PL) is a natural alkaloid isolated from the long pepper, Piper longum L., and has shown substantial cancer-preventive and therapeutic efficacy against variety of cancers. However, delivering its effective concentration in pancreatic tumors has been challenging. In this study, we have synthesized and characterized a novel nano-formulation of PL composed of a PLGA core (PLGA-PL), stabilized with polyvinyl alcohol (PVA) and coated with poly-L-lysine (PLL), and evaluated its therapeutic effects against PanCa. Methods: The various physicochemical approaches (FT-IR, DSC, TEM, TGA, and HPLC) was used to characterize the PLGA-PL formulation for particle size, chemical composition, and drug loading efficiency. Cellular uptake of PLGA-PL was achieved in incubation with PLGA-PL in PanCa cells. Further, the therapeutic efficacy of PLGA-PL was determined by using various in vitro assays (MTS, wound healing, boyden chamber, cell cycle and apoptosis assays) using PanCa cells. The effects of PLGA-PL on various key oncogenic signaling pathways were evaluated by qRT-PCR, Western blot, confocal microscopy, immunohistochemistry (IHC) analyses. Results: Our novel PLGA-PL formulation has an average size of 110 nm in dynamic light scattering and a zeta potential range of -6.52 to -7.68 mV with excellent PL loading efficiency. Cellular uptake and internalization studies show that PLGA-PL escapes lysosomal degradation, allowing for effective endosomal release into the cytosol. PLGA-PL showed superior anti-cancer activity in various PanCa cells (BxPC-3, HPAF-II, AsPC-1, Panc-1, and MIA PaCa-2) compared to free PL. Moreover, PLGA-PL showed a remarkable inhibition of the migration and invasion potential of PanCa cells. Furthermore, PLGA-PL more effectively inhibited the components of the Shh pathway and Gli targets as determined by qPCR and Western blot analysis. Additionally, PLGA-PL treatment targets cancer stem cells by regulating pluripotency, maintaining stemness factors (Oct-4, Sox2, Nanog and c-Myc), and limiting tumor sphere formation. Conclusions: Taken together, our results demonstrate that PLGA-PL nanoformulation exhibits superior anti-cancer potential than free PL against and could be used as a novel therapeutic modality for the management of PanCa. Citation Format: Vivek Kumar Kashyap, Godwin P. Darkwah, Swati Dhasmana, Neeraj Chauhan, Anupam Dhasmana, Sudhir Kotnala, Partha Laskar, Mohammed Sikander, Bilal B. Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Novel nanoformulation of piperlongumine for pancreatic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 305.