Emerging Trends in Gastrointestinal Cancer Targeted Therapies: Harnessing Tumor Microenvironment, Immune Factors, and Metabolomics Insights.

Gastrointestinal (GI) cancers are the leading cause of new cancer cases and cancer-related deaths worldwide. The treatment strategies for patients with GI tumors have focused on oncogenic molecular profiles associated with tumor cells. Recent evidence has demonstrated that the tumor cell functions are modulated by its microenvironment, compromising fibroblasts, extracellular matrices, microbiome, immune cells, and the enteric nervous system. Along with the tumor microenvironment components, alterations in key metabolic pathways have emerged as a hallmark of tumor cells. From these perspectives, this review will highlight the functions of different cellular components of the GI tumor microenvironment and their implications for treatment. Furthermore, we discuss the major metabolic reprogramming in GI tumor cells and how understanding metabolic rewiring could lead to new therapeutic strategies. Finally, we briefly summarize the targeted agents currently being studied in GI cancers. Understanding the complex interplay between tumor cell-intrinsic and -extrinsic factors during tumor progression is critical for developing new therapeutic strategies.

Cancer-Associated Fibroblast Induces Acinar-to-Ductal Cell Transdifferentiation and Pancreatic Cancer Initiation Via LAMA5/ITGA4 Axis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. METHODS: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs). RESULTS: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation. CONCLUSIONS: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception.

Chemokine-mucinome interplay in shaping the heterogeneous tumor microenvironment of pancreatic cancer.

Pancreatic cancer (PC) is exemplified by a complex immune-suppressive, fibrotic tumor microenvironment (TME), and aberrant expression of mucins. The constant crosstalk between cancer cells, cancer-associated fibroblasts (CAFs), and the immune cells mediated by the soluble factors and inflammatory mediators including cytokines, chemokines, reactive oxygen species (ROS) promote the dynamic temporal switch towards an immune-escape phenotype in the neoplastic cells and its microenvironment that bolsters disease progression. Chemokines have been studied in PC pathogenesis, albeit poorly in the context of mucins, tumor glycocalyx, and TME heterogeneity (CAFs and immune cells). With correlative analysis from PC patients' transcriptome data, support from available literature, and scientific arguments-based speculative extrapolations in terms of disease pathogenesis, we have summarized in this review a comprehensive understanding of chemokine-mucinome interplay during stromal modulation and immune-suppression in PC. Future studies should focus on deciphering the complexities of chemokine-mediated control of glycocalyx maturation, immune infiltration, and CAF-associated immune suppression. Knowledge extracted from such studies will be beneficial to mechanistically correlate the mucin-chemokine abundance in serum versus pancreatic tumors of patients, which may aid in prognostication and stratification of PC patients for immunotherapy.

Mucin 5AC Serves as the Nexus for ß-Catenin/c-Myc Interplay to Promote Glutamine Dependency During Pancreatic Cancer Chemoresistance.

BACKGROUND & AIMS: A major clinical challenge for patients with pancreatic cancer (PC) is metabolic adaptation. Neoplastic cells harboring molecular perturbations suffice for their increased anabolic demand and nucleotide biosynthesis to acquire chemoresistance. The mucin 5AC expressed de novo in malignant pancreas promotes cancer cell stemness and is significantly associated with poor patient survival. Identification of MUC5AC-associated drivers of chemoresistance through metabolic alterations may facilitate the sculpting of a new combinatorial regimen. METHODS: The contributions of MUC5AC to glutaminolysis and gemcitabine resistance were examined by The Cancer Genome Atlas data analysis, RNA sequencing, and immunohistochemistry analysis on pancreatic tissues of KrasG12D;Pdx1-Cre (KC) and KrasG12D;Pdx1-Cre;Muc5ac-/- mice. These were followed by metabolite flux assays as well as biochemical and xenograft studies on MUC5AC-depleted human and murine PC cells. Murine and human pancreatic 3-dimensional tumoroids were used to evaluate the efficacy of gemcitabine in combination with ß-catenin and glutaminolysis inhibitors. RESULTS: Transcriptional analysis showed that high MUC5AC-expressing human and autochthonous murine PC tumors exhibit higher resistance to gemcitabine because of enhanced glutamine use and nucleotide biosynthesis. Gemcitabine treatment led to MUC5AC overexpression, resulting in disruption of E-cadherin/ß-catenin junctions and the nuclear translocation of ß-catenin, which increased c-Myc expression, with a concomitant rise in glutamine uptake and glutamate release. MUC5AC depletion and glutamine deprivation sensitized human PC cells to gemcitabine, which was obviated by glutamine replenishment in MUC5AC-expressing cells. Coadministration of ß-catenin and glutaminolysis inhibitors with gemcitabine abrogated the MUC5AC-mediated resistance in murine and human tumoroids. CONCLUSIONS: The MUC5AC/ß-catenin/c-Myc axis increases the uptake and use of glutamine in PC cells, and cotargeting this axis along with gemcitabine may improve therapeutic efficacy in PC.

Malondialdehyde-Acetaldehyde Extracellular Matrix Protein Adducts Attenuate Unfolded Protein Response During Alcohol and Smoking-Induced Pancreatitis.

BACKGROUND & AIMS: Epidemiological studies have established alcohol and smoking as independent risk factors for recurrent acute pancreatitis and chronic pancreatitis. However, the molecular players responsible for the progressive loss of pancreatic parenchyma and fibroinflammatory response are poorly characterized. METHODS: Tandem mass tag-based proteomic and bioinformatics analyses were performed on the pancreata of mice exposed to alcohol, cigarette smoke, or a combination of alcohol and cigarette smoke. Biochemical, immunohistochemistry, and transcriptome analyses were performed on the pancreatic tissues and primary acinar cells treated with cerulein in combination with ethanol (50 mmol/L) and cigarette smoke extract (40 µg/mL) for the mechanistic studies. RESULTS: A unique alteration in the pancreatic proteome was observed in mice exposed chronically to the combination of alcohol and cigarette smoke (56.5%) compared with cigarette smoke (21%) or alcohol (17%) alone. The formation of toxic metabolites (P < .001) and attenuated unfolded protein response (P < .04) were the significantly altered pathways on combined exposure. The extracellular matrix (ECM) proteins showed stable malondialdehyde-acetaldehyde (MAA) adducts in the pancreata of the combination group and chronic pancreatitis patients with a history of smoking and alcohol consumption. Interestingly, MAA-ECM adducts significantly suppressed expression of X-box-binding protein-1, leading to acinar cell death in the presence of alcohol and smoking. The stable MAA-ECM adducts persist even after alcohol and smoking cessation, and significantly delay pancreatic regeneration by abrogating the expression of cyclin-dependent kinases (CDK7 and CDK5) and regeneration markers. CONCLUSIONS: The combined alcohol and smoking generate stable MAA-ECM adducts that increase endoplasmic reticulum stress and acinar cell death due to attenuated unfolded protein response and suppress expression of cell cycle regulators. Targeting aldehyde adducts might provide a novel therapeutic strategy for the management of recurrent acute pancreatitis and chronic pancreatitis.

Mucins reprogram stemness, metabolism and promote chemoresistance during cancer progression.

Mucins are high-molecular-weight glycoproteins dysregulated in aggressive cancers. The role of mucins in disease progression, tumor proliferation, and chemotherapy resistance has been studied extensively. This article provides a comprehensive review of mucin's function as a physical barrier and the implication of mucin overexpression in impeded drug delivery to solid tumors. Mucins regulate the epithelial to mesenchymal transition (EMT) of cancer cells via several canonical and non-canonical oncogenic signaling pathways. Furthermore, mucins play an extensive role in enriching and maintaining the cancer stem cell (CSC) population, thereby sustaining the self-renewing and chemoresistant cellular pool in the bulk tumor. It has recently been demonstrated that mucins regulate the metabolic reprogramming during oncogenesis and cancer progression, which account for tumor cell survival, proliferation, and drug-resistance. This review article focuses on delineating mucin's role in oncogenic signaling and aberrant regulation of gene expressions, culminating in CSC maintenance, metabolic rewiring, and development of chemoresistance, tumor progression, and metastasis.

Unraveling mucin domains in cancer and metastasis: when protectors become predators.

A dynamic mucosal layer shields the epithelial cells lining the body cavities and is made up of high molecular weight, heavily glycosylated, multidomain proteins called mucins. Mucins, broadly grouped into transmembrane and secreted mucins, are the first responders to any mechanical or chemical insult to the epithelia and help maintain tissue homeostasis. However, their intrinsic properties to protect and repair the epithelia are exploited during oncogenic processes, where mucins are metamorphosed to aid the tumor cells in their malignant journey. Diverse domains, like the variable number tandem repeats (VNTR), sea urchin sperm protein enterokinase and agrin (SEA), adhesion-associated domain (AMOP), nidogen-like domain (NIDO), epidermal growth factor-like domain (EGF), and von Willebrand factor type D domain (vWD) on mucins, including MUC1, MUC4, MUC5AC, MUC5B, and MUC16, have been shown to facilitate cell-to-cell and cell-to-matrix interactions, and cell-autonomous signaling to promote tumorigenesis and distant dissemination of tumor cells. Several obstacles have limited the study of mucins, including technical difficulties in working with these huge glycoproteins, the dearth of scientific tools, and lack of animal models; thus, the tissue-dependent and domain-specific roles of mucins during mucosal protection, chronic inflammation, tumorigenesis, and hematological dissemination of malignant cells are still unclear. Future studies should try to integrate information on the rheological, molecular, and biological characteristics of mucins to comprehensively delineate their pathophysiological role and evaluate their suitability as targets in future diagnostic and therapeutic strategies.

RNA Polymerase II-Associated Factor 1 Regulates Stem Cell Features of Pancreatic Cancer Cells, Independently of the PAF1 Complex, via Interactions With PHF5A and DDX3.

BACKGROUND & AIMS: It is not clear how pancreatic cancer stem cells (CSCs) are regulated, resulting in ineffective treatments for pancreatic cancer. PAF1, a RNA polymerase II-associated factor 1 complex (PAF1C) component, maintains pluripotency of stem cells, by unclear mechanisms, and is a marker of CSCs. We investigated mechanisms by which PAF1 maintains CSCs and contributes to development of pancreatic tumors. METHODS: Pancreatic cancer cell lines were engineered to knockdown PAF1 using inducible small hairpin RNAs. These cells were grown as orthotopic tumors in athymic nude mice and PAF1 knockdown was induced by administration of doxycycline in drinking water. Tumor growth and metastasis were monitored via IVIS imaging. CSCs were isolated from pancreatic cancer cell populations using flow cytometry and characterized by tumor sphere formation, tumor formation in nude mice, and expression of CSC markers. Isolated CSCs were depleted of PAF1 using the CRISPR/Cas9 system. PAF1-regulated genes in CSCs were identified via RNA-seq and PCR array analyses of cells with PAF1 knockdown. Proteins that interact with PAF1 in CSCs were identified by immunoprecipitations and mass spectrometry. We performed chromatin immunoprecipitation sequencing of CSCs to confirm the binding of the PAF1 sub-complex to target genes. RESULTS: Pancreatic cancer cells depleted of PAF1 formed smaller and fewer tumor spheres in culture and orthotopic tumors and metastases in mice. Isolated CSCs depleted of PAF1 downregulated markers of self-renewal (NANOG, SOX9, and ß-CATENIN), of CSCs (CD44v6, and ALDH1), and the metastasis-associated gene signature, compared to CSCs without knockdown of PAF1. The role of PAF1 in CSC maintenance was independent of its RNA polymerase II-associated factor 1 complex component identity. We identified DDX3 and PHF5A as proteins that interact with PAF1 in CSCs and demonstrated that the PAF1-PHF5A-DDX3 sub-complex bound to the promoter region of Nanog, whose product regulates genes that control stemness. Levels of the PAF1-DDX3 and PAF1-PHF5A were increased and co-localized in human pancreatic tumor specimens, human pancreatic tumor-derived organoids, and organoids derived from tumors of KPC mice, compared with controls. Binding of DDX3 and PAF1 to the Nanog promoter, and the self-renewal capacity of CSCs, were decreased in cells incubated with the DDX3 inhibitor RK-33. CSCs depleted of PAF1 downregulated genes that regulate stem cell features (Flot2, Taz, Epcam, Erbb2, Foxp1, Abcc5, Ddr1, Muc1, Pecam1, Notch3, Aldh1a3, Foxa2, Plat, and Lif). CONCLUSIONS: In pancreatic CSCs, PAF1 interacts with DDX3 and PHF5A to regulate expression of NANOG and other genes that regulate stemness. Knockdown of PAF1 reduces the ability of orthotopic pancreatic tumors to develop and progress in mice and their numbers of CSCs. Strategies to target the PAF1-PHF5A-DDX3 complex might be developed to slow or inhibit progression of pancreatic cancer.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1.

BACKGROUND & AIMS: Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. METHODS: KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. RESULTS: Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. CONCLUSIONS: Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

Novel role of O-glycosyltransferases GALNT3 and B3GNT3 in the self-renewal of pancreatic cancer stem cells.

BACKGROUND: Glycosylation plays a critical role in the aggressiveness of pancreatic cancer (PC). Emerging evidences indicate significant involvement of cancer stem cells (CSCs) in PC aggressiveness. However, the importance of glycosylation in pancreatic cancer stem cells (PCSCs) is yet to be addressed. Hence, we evaluated the potential role of glycosylation in maintenance of stemness of PCSCs. METHODS: Effect of glycosylation specific inhibitors on growth and PCSCs of PC cells was assessed by MTT assay and Side Population (SP) analysis. Isolated PCSCs/SP were characterized using molecular and functional assays. Expression of tumor-associated carbohydrate antigens (TACAs) was analyzed in PCSCs by western blotting. Effect of tunicamycin on PCSCs was analyzed by tumorsphere, clonogenicity, migration assay and immunoblotting for CSCs markers. The differential expression of glycogenes in PCSCs compared to non-CSCs were determined by RT-qPCR, immunoblotting and immunofluorescence. Co-expression of GALNT3 and B3GNT3 with CD44v6 was assessed in progression stages of KrasG12D; Pdx-1-Cre (KC) and KrasG12D; p53R172H; Pdx-1-Cre (KPC) tumors by immunofluorescence. Transient and CRISPR/Cas9 silencing of GALNT3 and B3GNT3 was performed to examine their effect on CSCs maintenance. RESULTS: Inhibition of glycosylation decreased growth and CSCs/SP in PC cells. PCSCs overexpressed CSC markers (CD44v6, ESA, SOX2, SOX9 and ABCG2), exhibited global expressional variation of TACAs and showed higher self-renewal potential. Specifically, N-glycosylation inhibition, significantly decreased tumorsphere formation, migration, and clonogenicity of PCSCs, as well as hypo-glycosylated CD44v6 and ESA. Of note, glycosyltransferases (GFs), GALNT3 and B3GNT3, were significantly overexpressed in PCSCs and co-expressed with CD44v6 at advanced PDAC stages in KC and KPC tumors. Further, GALNT3 and B3GNT3 knockdown led to a decrease in the expression of cell surface markers (CD44v6 and ESA) and self-renewal markers (SOX2 and OCT3/4) in PCSCs. Interestingly, CD44v6 was modified with sialyl Lewis a in PCSCs. Finally, CRISPR/Cas9-mediated GALNT3 KO significantly decreased self-renewal, clonogenicity, and migratory capacity in PCSCs. CONCLUSIONS: Taken together, for the first time, our study showed the importance of glycosylation in mediating growth, stemness, and maintenance of PCSCs. These results indicate that elevated GALNT3 and B3GNT3 expression in PCSCs regulate stemness through modulating CSC markers.

Lupeol evokes anticancer effects in oral squamous cell carcinoma by inhibiting oncogenic EGFR pathway.

Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays. Lupeol-mediated induction of apoptosis was examined by caspase 3/7 assay and flow cytometry. Effect of Lupeol on EGFR in the presence or absence of EGF was delineated by Western blot. The mRNA stability assay was performed to check the role of Lupeol on COX-2 mRNA regulation. Lupeol inhibited proliferation of OSCC cells in vitro by inducing apoptosis 48 h post treatment. Ligand-induced phosphorylation of EGFR and subsequent activation of its downstream molecules such as protein kinase B (PKB or AKT), I kappa B (IκB), and nuclear factor kappa B (NF-κB) was also found to be, in part, suppressed. Interestingly, Lupeol suppressed expression of COX-2 at mRNA and protein level in a time-dependent manner. Primary explants from oral squamous cell carcinoma tissues further confirmed significant inhibition of proliferation (Ki67) in Lupeol-treated explants as compared to untreated control at 48 h. Together these data suggest that Lupeol may act as a potent inhibitor of the EGFR signaling in OSCC and therefore imply its role in triggering antitumor efficacy.

Connectivity mapping-based identification of pharmacological inhibitor targeting HDAC6 in aggressive pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials.

Elevated PAF1-RAD52 axis confers chemoresistance to human cancers.

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies.

Aberrantly expressed onco-mucin 16 (MUC16) and its post-cleavage generated surface tethered carboxy-terminal (MUC16-Cter) domain are strongly associated with poor prognosis and lethality of pancreatic (PC) and non-small cell lung cancer (NSCLC). To date, most anti-MUC16 antibodies are directed towards the extracellular domain of MUC16 (CA125), which is usually cleaved and shed in the circulation hence obscuring antibody accessibility to the cancer cells. Herein, we establish the utility of targeting a post-cleavage generated, surface-tethered oncogenic MUC16 carboxy-terminal (MUC16-Cter) domain by using a novel chimeric antibody in human IgG1 format, ch5E6, whose epitope expression directly correlates with disease severity in both cancers. ch5E6 binds and interferes with MUC16-associated oncogenesis, suppresses the downstream signaling pFAK(Y397)/p-p70S6K(T389)/N-cadherin axis and exert antiproliferative effects in cancer cells, 3D organoids, and tumor xenografts of both PC and NSCLC. The robust clinical correlations observed between MUC16 and N-cadherin in patient tumors and metastatic samples imply ch5E6 potential in targeting a complex and significantly occurring phenomenon of epithelial to mesenchymal transition (EMT) associated with disease aggressiveness. Our study supports evaluating ch5E6 with standard-of-care drugs, to potentially augment treatment outcomes in malignancies inflicted with MUC16-associated poor prognosis.

PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer.

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

MUC16 Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma by Upregulating NRP2-Associated Cell Adhesion.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, as it commonly metastasizes to the liver resulting in an overall poor prognosis. However, the molecular mechanism involved in liver metastasis remains poorly understood. Here, we aimed to identify the MUC16-mediated molecular mechanism of PDAC-liver metastasis. Previous studies demonstrated that MUC16 and its C-terminal (Cter) domain are involved in the aggressiveness of PDAC. In this study, we observed MUC16 and its Cter expression significantly high in human PDAC tissues, PDAC organoids, and metastatic liver tissues, while no expression was observed in normal pancreatic tissues using IHC and immunofluorescence (IFC) analyses. MUC16 knockdown in SW1990 and CD18/HPAF PDAC cells significantly decreased the colony formation, migration, and endothelial/p-selectin binding. In contrast, MUC16-Cter ectopic overexpression showed significantly increased colony formation and motility in MiaPaCa2 pancreatic cancer cells. Interestingly, MUC16 promoted cell survival and colonization in the liver, mimicking an ex vivo environment. Furthermore, MUC16 enhanced liver metastasis in the in vivo mouse model. Our integrated analyses of RNA-sequencing suggested that MUC16 alters Neuropilin-2 (NRP2) and cell adhesion molecules in pancreatic cancer cells. Furthermore, we identified that MUC16 regulated NRP2 via JAK2/STAT1 signaling in PDAC. NRP2 knockdown in MUC16-overexpressed PDAC cells showed significantly decreased cell adhesion and migration. Overall, the findings indicate that MUC16 regulates NRP2 and induces metastasis in PDAC. IMPLICATIONS: This study shows that MUC16 plays a critical role in PDAC liver metastasis by mediating NRP2 regulation by JAK2/STAT1 axis, thereby paving the way for future therapy efforts for metastatic PDAC.

Small molecule inhibitor against onco-mucins disrupts Src/FosL1 axis to enhance gemcitabine efficacy in pancreatic ductal adenocarcinoma.

Mucin MUC4 is an aberrantly expressed oncogene in pancreatic ductal adenocarcinoma (PDAC), yet no pharmacological inhibitors have been identified to target MUC4. Here, we adapted an in silico screening method using the Cancer Therapeutic Response Database (CTRD) to Identify Small Molecule Inhibitors against Mucins (SMIMs). We identified Bosutinib as a candidate drug to target oncogenic mucins among 126 FDA-approved drugs from CTRD screening. Functionally, Bosutinib treatment alone/and in combination with gemcitabine (Gem)/5' fluorouracil (5FU) reduced in vitro viability, migration, and colony formation in multiple PDAC cell lines as well as human PDAC organoid prolifertaion and growth and in vivo xenograft growth. Further, biochemical and molecular analyses showed that Bosutinib exhibited these functional effects by downregulating MUC4 mucin at both transcript and translation levels in a dose- and time-dependent manner. Mechanistically, global transcriptome analysis in PDAC cells upon treatment with Bosutinib revealed disruption of the Src-ERK/AKT-FosL1 pathway, leading to decreased expression of MUC4 and MUC5AC mucins. Taken together, Bosutinib is a promising, novel, and highly potent SMIMs to target MUC4/MUC5AC mucins. This mucin-targeting effect of Bosutinib can be exploited in the future with cytotoxic agents to treat mucinous tumors.

Muc16 depletion diminishes KRAS-induced tumorigenesis and metastasis by altering tumor microenvironment factors in pancreatic ductal adenocarcinoma.

MUC16, membrane-bound mucin, plays an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). However, the pathological role of MUC16 in the PDAC progression, tumor microenvironment, and metastasis in cooperation with KrasG12D and Trp53R172H mutations remains unknown. Deletion of Muc16 with activating mutations KrasG12D/+ and Trp53R172H/+ in mice significantly decreased progression and prolonged overall survival in KrasG12D/+; Trp53R172H/+; Pdx-1-Cre; Muc16-/- (KPCM) and KrasG12D/+; Pdx-1-Cre; Muc16-/- (KCM), as compared to KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) and KrasG12D/+; Pdx-1-Cre (KC) mice, respectively. Muc16 knockout pancreatic tumor (KPCM) displays decreased tumor microenvironment factors and significantly reduced incidence of liver and lung metastasis compared to KPC. Furthermore, in silico data analysis showed a positive correlation of MUC16 with activated stroma and metastasis-associated genes. KPCM mouse syngeneic cells had significantly lower metastatic and endothelial cell binding abilities than KPC cells. Similarly, KPCM organoids significantly decreased the growth rate compared to KPC organoids. Interestingly, RNA-seq data revealed that the cytoskeletal proteins Actg2, Myh11, and Pdlim3 were downregulated in KPCM tumors. Further knockdown of these genes showed reduced metastatic potential. Overall, our results demonstrate that Muc16 alters the tumor microenvironment factors during pancreatic cancer progression and metastasis by changing the expression of Actg2, Myh11, and Pdlim3 genes.

Recent advances in organoid development and applications in disease modeling.

An improved understanding of stem cell niches, organogenesis, and disease models has paved the way for developing a three-dimensional (3D) organoid culture system. Organoid cultures can be derived from primary tissues (single cells or tissue subunits), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs). As a significant technological breakthrough, 3D organoid models offer a promising approach for understanding the complexities of human diseases ranging from the mechanistic investigation of disease pathogenesis to therapy. Here, we discuss the recent applications, advantages, and limitations of organoids as in vitro models for studying metabolomics, drug development, infectious diseases, and the gut microbiome. We further discuss the use of organoids in cancer modeling using high throughput sequencing approaches.

The Current Landscape of Antibody-based Therapies in Solid Malignancies.

Over the past three decades, monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy. Still, this benefit remains restricted to a small proportion of patients due to moderate response rates and resistance emergence. The field has started to embrace better mAb-based formats with advancements in molecular and protein engineering technologies. The development of a therapeutic mAb with long-lasting clinical impact demands a prodigious understanding of target antigen, effective mechanism of action, gene engineering technologies, complex interplay between tumor and host immune system, and biomarkers for prediction of clinical response. This review discusses the various approaches used by mAbs for tumor targeting and mechanisms of therapeutic resistance that is not only caused by the heterogeneity of tumor antigen, but also the resistance imposed by tumor microenvironment (TME), including inefficient delivery to the tumor, alteration of effector functions in the TME, and Fc-gamma receptor expression diversity and polymorphism. Further, this article provides a perspective on potential strategies to overcome these barriers and how diagnostic and prognostic biomarkers are being used in predicting response to mAb-based therapies. Overall, understanding these interdependent parameters can improve the current mAb-based formulations and develop novel mAb-based therapeutics for achieving durable clinical outcomes in a large subset of patients.