Mesothelin Enhances Tumor Vascularity in Newly Forming Pancreatic Peritoneal Metastases.

Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mesothelin (MSLN). Overexpression or knockdown of MSLN has been implicated in PDAC aggressiveness. This activity has been ascribed to MSLN-induced activation of MAPK or NF-κB signaling pathways and to interaction of MSLN with its only known binding partner, MUC16. Here, we used CRISPR/Cas9 gene editing to delete MSLN from PDAC, then restored expression of wild-type (WT) or Y318A mutant MSLN by viral transduction. We found that MSLN KO cells grew in culture and as subcutaneous tumors in mouse xenografts at the same rate as WT cells but formed intraperitoneal metastases poorly. Complementation with WT MSLN restored intraperitoneal growth, whereas complementation with Y318A mutant MSLN, which does not bind MUC16, was ineffective at enhancing growth in both MUC16(+) and MUC16(-) models. Restoration of WT MSLN did enhance growth but did not affect cell-to-cell binding, cell viability in suspension or signaling pathways previously identified as contributing to the protumorigenic effect of MSLN. RNA deep sequencing of tumor cells identified no changes in transcriptional profile that could explain the observed phenotype. Furthermore, no histologic changes in tumor cell proliferation or morphology were observed in mature tumors. Examination of nascent MSLN KO tumors revealed decreased microvascular density as intraperitoneal tumors were forming, followed by decreased proliferation, which resolved by 2 weeks postimplantation. These data support a model whereby MSLN expression by tumor cells contributes to metastatic colonization. IMPLICATIONS: MSLN confers a growth advantage to tumor cells during colonization of peritoneal metastasis. Therapeutic blockade of MSLN might limit peritoneal spread.

Protein Synthesis Inhibition Activity of Mesothelin Targeting Immunotoxin LMB-100 Decreases Concentrations of Oncogenic Signaling Molecules and Secreted Growth Factors.

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24⁻48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endothelial Growth Factor (VEGF) following treatment with cytostatic LMB-100 doses both in culture and in mouse tumors. Decreased VEGF concentration did not change tumor vasculature density, however, LMB-100 caused tissue-specific changes in concentrations of secreted factors made by non-cancer cells. In summary, our data indicate that PSI caused by cytostatic LMB-100 doses preferentially depletes short-lived proteins such as oncogenic signaling molecules and CCSGFs.