Abstract NG12: Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression
Authors: Pitarresi Jason Robert 
Published: April 2023

Abstract
Purpose: Metastasis is the leading cause of cancer-related death in pancreatic ductal adenocarcinoma (PDAC), yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by the PTHLH gene) is located directly adjacent to KRAS and is co-amplified as part of the KRAS amplicon in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP, and downstream activation of calcium signaling, exerts its own oncogenic and pro-metastatic phenotypes beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Finally, we postulate that PTHrP-mediated Calcium signaling may be responsible for driving other non-cell autonomous PDAC-associated phenotypes, including cancer cachexia and the organization of an immunosuppressive microenvironment.
Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine PTHrP in the autochthonous KPCY model of pancreatic cancer (mutant Kras and mutant p53 activated in the pancreatic epithelium). To functionally demonstrate the pro-metastatic and cachexia-inducing roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection and mouse hospital pre-clinical trials.
Results: In silico analysis established that PTHLH (the gene encoding the PTHrP protein) is co-amplified along with KRAS in TCGA and correlates with significantly decreased overall survival. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Mechanistic studies revealed that intracellular calcium was responsible for turning on an epithelial-to-mesenchymal (EMT) program that could be impeded by inhibiting PTHrP, thus blocking tumor cells from entering the metastatic cascade. Additionally, both genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia and metastasis observed upon orthotopic implantation. Thus, PTHrP is both necessary and sufficient to induce cachexia and metastasis. Additionally, mice implanted with PTHrP overexpressing tumor cells had a dramatic reduction in overall survival. Finally, we demonstrated that PTHrP is responsible for organizing a pro-tumor immunosuppressive microenvironment by recruiting Myeloid Derived Suppressive Cells (MDSCs). Blocking PTHrP signaling reduces the degree of local immune suppression allowing for the enhanced recruitment of CD8+ T cells. Thus, PTHrP is a pleiotropic molecule that governs many pancreatic cancer-associated disease phenotypes.
Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving diverse pancreatic cancer phenotypes from metastasis to cachexia to immunosuppression, and future studies will look to translate anti-PTHrP therapy into clinical trials.
Citation Format: Jason Robert Pitarresi. Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression. [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 NG12.