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Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

ABSTRACT: Peritoneum is the second most common site of metastasis in patients with pancreatic ductal adenocarcinoma (PDAC). Peritoneal colonization is impaired in PDAC cells with knock-out (KO) of the cancer surface antigen mesothelin (MSLN) or by introducing Y318A mutation in MSLN to prevent binding to mucin-16 (MUC-16). MSLN has a membrane-bound form but is also shed to release soluble MSLN (sMSLN). Their individual roles in peritoneal metastasis are unknown. Here, a C-terminal truncated MSLN mutant (∆591) incapable of cell membrane insertion but proficient in secretion was engineered. Expression of ∆591 MSLN failed to rescue peritoneal metastasis in MSLN KO cells and inhibited peritoneal colonization when overexpressed in WT PDAC cells. Exposing PDAC cells to conditioned medium (CM) containing excess sMSLN impaired cancer cell clustering in vitro and in peritoneal fluid in vivo, while CM containing only Y318A sMSLN did not. These data demonstrate that interaction of membrane-bound MSLN with MUC-16 promotes cell clustering that is critical for efficient peritoneal metastasis. However, peritoneal colonization by MSLN KO cells was rescued by expression of ∆591 mutant MSLN bearing Y318A mutation, suggesting that sMSLN also has a MUC-16-independent role in peritoneal spread. Alterations in inflammatory signaling pathways occurred following KO cell exposure to CM containing sMSLN, and CM from cancer cells with intact peritoneal metastasis provoked increased KO cell secretion of IL-1α. While excess sMSLN inhibits cell clustering and peritoneal colonization, sMSLN may also promote PDAC peritoneal metastasis independent of MUC-16.

ORGANISM(S): Homo sapiens

PROVIDER: GSE260587 | GEO | 2024/12/19

REPOSITORIES: GEO

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Project description:Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous cancer in which differences in survival rates might be related to a variety in gene expression profiles. Although the molecular biology of PDAC begins to be revealed, genes or pathways that specifically drive tumour progression or metastasis are not well understood. Therefore, we performed microarray analyses on whole-tumour samples of 2 human PDAC subpopulations with similar clinicopathological features, but extremely distinct survival rates after potentially curative surgery, i.e., good outcome (OS and DFSM-bM-^@M-^I>M-bM-^@M-^I50M-bM-^@M-^Imonths) versus bad outcome (OSM-bM-^@M-^I<M-bM-^@M-^I19M-bM-^@M-^Imonths and DFSM-bM-^@M-^I<M-bM-^@M-^I7M-bM-^@M-^Imonths). Additionally, liver- and peritoneal metastases were analysed and compared to primary cancer tissue. The integrin and ephrin receptor families were upregulated in all PDAC samples, irrespective of outcome, supporting an important role of the interaction between pancreatic cancer cells and the surrounding desmoplastic reaction in tumorigenesis and cancer progression. Moreover, some components, such as ITGB1 and EPHA2, were upregulated in PDAC samples with a poor outcome, Additionally, overexpression of the non-canonical Wnt/M-NM-2-catenin pathway and EMT genes in PDAC samples with bad versus good outcome suggests their contribution to the invasiveness of pancreatic cancer, with M-NM-2-catenin being also highly upregulated in metastatic tissue. Thus, we conclude that components of the integrin and ephrin pathways and EMT-related genes might serve as molecular markers in pancreatic cancer as their expression seems to be related with prognosis. Microarray analysis was performed on 'Good' and 'Bad' patient samples (samples with similar pathological characteristics were chosen based on the definition of the 2 diverse survival outcome groups and the required RIN values above 7.1), on surrounding non-tumoural pancreatic control samples, liver metastasis (LM) and peritoneal metastasis (PM). Comparisons between good vs. control, bad vs. control, good vs. bad, and primary pancreatic cancer versus metastasis were performed.

Project description:Pancreatic ductal adenocarcinoma (PDAC) remains among the most lethal of human cancers underscoring the need to identify new therapeutic vulnerabilities. Previously, we reported high expression of transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 in human PDAC, however their precise role in PDAC formation or progression remains unclear. Here, we have studied PDAC tumor dependency on CtBPs for growth and metastasis using an orthotopic syngeneic pancreatic tumor mouse model. CRISPR-based homozygous deletion of Ctbp2 dramatically decreased PDAC tumor growth, drastically reduced metastatic potential, and significantly prolonged survival. Interrogating differential gene expression of the orthotopic PDAC tumors from CtBP2 WT vs. CtBP2 KO cohorts, we identified significant downregulation of the EGFR-superfamily receptor ErbB3 in CtBP2 KO tumors, and further determined that CtBP regulates expression of both the ErbB2 and 3 EGFR superfamily genes in PDAC cells. We therefore hypothesized that CtBP regulation of physiologic ErbB2/3 signaling contributes, in part, to PDAC growth and metastasis. Indeed, we observed that CtBP2 KO cells exhibited severely attenuated activation of phospho-Akt after neuregulin stimulation of ErbB2/3, and we demonstrate that human PDAC cells also show CtBP dependence of ErbB2/3 expression. Our results suggest that a subset of PDAC tumors is dependent on physiologic ErbB2/3 signaling and could be targeted by pharmacologic inhibitors of ErbB2 and/or ErbB3. Providing proof of concept, the ErbB2-targeted multikinase inhibitor lapatinib, but not the ErbB1/EGFR targeted agent erlotinib, effectively killed ErbB3 expressing PDAC cells, while CtBP2 KO cells where ErbB3 expression was extinguished, were resistant to lapatinib. Taken together, our data suggests that ErBb2/3 targeted therapeutics can effectively target a critical PDAC dependency on physiologic ErbB2/3 signaling which is the result of CtBP’s oncogenic transcriptional program that drives PDAC tumor progression.

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Excess shed mesothelin disrupts pancreatic cancer cell clustering to impair peritoneal colonization

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