Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS: HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

Targeting TNF-α-producing macrophages activates antitumor immunity in pancreatic cancer via IL-33 signaling.

Pancreatic ductal adenocarcinoma (PDA) remains resistant to immune therapies, largely owing to robustly fibrotic and immunosuppressive tumor microenvironments. It has been postulated that excessive accumulation of immunosuppressive myeloid cells influences immunotherapy resistance, and recent studies targeting macrophages in combination with checkpoint blockade have demonstrated promising preclinical results. Yet our understanding of tumor-associated macrophage (TAM) function, complexity, and diversity in PDA remains limited. Our analysis reveals significant macrophage heterogeneity, with bone marrow-derived monocytes serving as the primary source for immunosuppressive TAMs. These cells also serve as a primary source of TNF-α, which suppresses expression of the alarmin IL-33 in carcinoma cells. Deletion of Ccr2 in genetically engineered mice decreased monocyte recruitment, resulting in profoundly decreased TNF-α and increased IL-33 expression, decreased metastasis, and increased survival. Moreover, intervention studies targeting CCR2 with a new orthosteric inhibitor (CCX598) rendered PDA susceptible to checkpoint blockade, resulting in reduced metastatic burden and increased survival. Our data indicate that this shift in antitumor immunity is influenced by increased levels of IL-33, which increases dendritic cell and cytotoxic T cell activity. These data demonstrate that interventions to disrupt infiltration of immunosuppressive macrophages, or their signaling, have the potential to overcome barriers to effective immunotherapeutics for PDA.

Antifibrotic Therapy Disrupts Stromal Barriers and Modulates the Immune Landscape in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest forms of cancer, in part, because it is largely refractory to current therapies. The failure of most standard therapies in PDA, as well as promising immune therapies, may be largely ascribed to highly unique and protective stromal microenvironments that present significant biophysical barriers to effective drug delivery, that are immunosuppressive, and that can limit the distribution and function of antitumor immune cells. Here, we utilized stromal reengineering to disrupt these barriers and move the stroma toward normalization using a potent antifibrotic agent, halofuginone. In an autochthonous genetically engineered mouse model of PDA, halofuginone disrupted physical barriers to effective drug distribution by decreasing fibroblast activation and reducing key extracellular matrix elements that drive stromal resistance. Concomitantly, halofuginone treatment altered the immune landscape in PDA, with greater immune infiltrate into regions of low hylauronan, which resulted in increased number and distribution of both classically activated inflammatory macrophages and cytotoxic T cells. In concert with a direct effect on carcinoma cells, this led to widespread intratumoral necrosis and reduced tumor volume. These data point to the multifunctional and critical role of the stroma in tumor protection and survival and demonstrate how compromising tumor integrity to move toward a more normal physiologic state through stroma-targeting therapy will likely be an instrumental component in treating PDA. SIGNIFICANCE: This work demonstrates how focused stromal re-engineering approaches to move toward normalization of the stroma disrupt physical barriers to effective drug delivery and promote antitumor immunity.See related commentary by Huang and Brekken, p. 328.