Search

Latent recurrence following curative-intent pancreatic cancer surgery is a major clinical problem thought to be due to the reactivation of dormant tumor cells that disseminate before the primary tumor has been removed. How dormancy is established and ultimately reversed to drive recurrence is poorly understood. Here we introduce a novel mouse model of pancreatic cancer dormancy in which we establish distal pancreatic tumors by orthotopic injection, resect them four weeks later and then follow the mice for recurrence. We observed recurrence patterns and survival outcomes that mimic human patients undergoing surgery for pancreatic cancer where two thirds of the mice succumb to early metastatic recurrence (median survival 26 days) and one third of the mice (called dormant mice) live substantially long (median survival 554 days) without clinic evidence of disease yet harbor disseminated tumor cells in most organs of the body. Disseminated tumor cells isolated from the livers of dormant mice were quiescent, exhibited stem cell properties and upregulated the expression of Dec2, a circadian rhythm gene. Overexpression of Dec2 induced quiescence suggesting that it may be a driver of the dormant phenotype. Curiously, endogenous Dec2 actually increased the growth and metastasis of nondormant tumors. Dec2WT and Dec2 knockout cell lines were used to generate resectable orthotopic tumors in immune-competent mice, and loss of Dec2 decreased tumor growth, dissemination and metastasis, and the median survival of mice with DecWT tumors was 25 days whereas 70% of the mice with the otherwise isogenic Dec2KOs were still alive after 200 days. Notably, these differences in tumor size, dissemination, metastasis and survival were abrogated in nude mice, indicating an immune mediated mechanism. Dec2WT and Dec2KO cell lines expressed the same total amount of the antigen-presenting protein MHC-1, but much more of the MHC-1 was present on the surface of Dec2KO cell lines than on the Dec2WT cell lines. MHC-1 requires peptide loading before transport to the cell surface, and we see evidence for reduced production of small peptides in the DecKO cells. For instance the half-life of luciferase, a very potent immunogen in mice, was reduced in the Dec2KO cells, potentially providing small peptides for MHC-1 biosynthesis. Protein turnover may be upregulated to compensate for increases in translation, as ribosomal genes are more highly expressed in Dec2KO cells, including many c-Myc pathway genes. Indeed, Western blot analysis revealed upregulation of c-Myc protein in Dec2KO cells. These data suggest that Dec2 plays a novel role in the immune system by decreasing MHC-1 cell surface expression through decreasing peptide generation, protein translation and turnover. Citation Format: Chris R. Harris, Crissy Dudgeon, Orjola Prela, Lan Wang, Anthony Casabianca, Juliana Cazarin de Menezes, Christina Davidson, Paula Vertino, Brian Altman, Darren Carpizo. DEC2, a circadian rhythm transcription factor, promotes growth, metastasis and dormancy by facilitating immune cell invasion through decreasing cell surface expression of MHC-1 in a novel model of pancreatic cancer dormancy [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 1287.

The axonal guidance receptor UNC5B is considered to be a tumor suppressor because it promotes apoptosis in the absence of its protein ligand, NTN1. But we find that UNC5B is not a tumor suppressor of pancreatic ductal adenocarcinomas (PDAC), and instead promotes metastasis. In TCGA data, UNC5B expression associates with poor patient outcome. In vivo, pancreatic cancer metastasis was completely eliminated by knocking out UNC5B from the genetically engineered KPC mouse model of PDACs, and was reduced upon knockout of UNC5B in a splenectomy model of liver metastasis. An inhibitor of NTN1, NP137, also reduced metastasis in vivo. In vitro, knockout of UNC5B from mesenchymal pancreatic cell lines was sufficient to reduce many metastatic traits including invasiveness, EMT, and aerobic glycolysis. YAP1 is a known target of UNC5B and was overexpressed in high UNC5B expressing cell lines, which were sensitive to the YAP1 inhibitor CA3. NP137 is currently in clinical trial for several tumor types and we have been investigating which patients might respond best to this drug. Using an antibody (D9M7Z) that recognizes an epitope in UNC5B that overlaps with the caspase-3 cleavage site that is critical for UNC5B's apoptotic functions, we found two isoforms in murine and human PDACs: one isoform that contains the caspase-3-containing epitope as well as a previously-unreported isoform that to lack it. Cells expressing the caspase-3 site were much more sensitive to NP137. We also discovered genetic alterations in patient samples that induce expression of UNC5B. For instance, UNC5B was recurrently amplified in PDACs and these amplifications were highly focal to the UNC5B locus; also, UNC5B expression was increased by mutations of ARID1A, a component of the SWI/SNF transcription complex that is recurrently mutated in pancreatic adenocarcinomas. ARID1A mutations caused cell lines to become mesenchymal, but the cells returned to an epithelial phenotype upon knockout of UNC5B. In current and future clinical trials, NP137 may particularly benefit patients with tumors that harbor mutations in ARID1A or ARID1B, focal amplifications of UNC5B, and/or high expression of the caspase-3-containing isoform of UNC5B. Citation Format: Chris R. Harris, Orjola Prela, Lan Wang, Anthony Casabianca, Wade Narrow, Zachary Sechrist, Tracy Withers, Cory Shields, Asra Asad, Aram Hezel, Darren Carpizo. UNC5B promotes EMT and metastasis of pancreatic adenocarcinomas and expresses different isoforms that impact sensitivity to Netrin-1 blockade [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 1286.

Axon guidance pathways have often been linked to tumorigenesis by informatics analyses, but their roles in tumorigenesis are not well understood at a functional level. Here we show that axon guidance receptor UNC5B and its ligand, NTN1, promote the metastasis of pancreatic ductal adenocarcinomas (PDACs) by reducing anoikis and by promoting a mesenchymal phenotype. In vivo, removal of two floxed UNC5B alleles eliminated PDAC metastasis in the genetically engineered KPC mouse model, and also reduced metastasis by xenografted PDAC cell lines. PDAC metastasis also decreased upon treatment of animals with NP137, an antibody that targets NTN1. In vitro, deletion of UNC5B from pancreatic adenocarcinoma cell lines reduced invasiveness, and was sufficient to convert PDAC cell lines from an epithelial to a mesenchymal state. UNC5B also increased YAP1 expression but decreased anoikis; anoikis could then be restored by treatment with the YAP1 inhibitor CA3. Clinically, UNC5B expression associated with poor outcomes in patients with pancreatic adenocarcinomas. UNC5B and NTN1 expression increased upon truncation of the SWI/SNF component ARID1A, which is recurrently mutated in PDACs. Highly focal amplifications of UNC5B were also observed in some patient samples, and were mutually exclusive with mutations in ARID1A or ARID1B. NP137 is currently in clinical trials for various cancers, and may benefit pancreatic adenocarcinoma patients, particularly those with UNC5B amplifications or with ARID1A/ARID1B mutations. Citation Format: Chris Harris, Anthony Casabianca, Zach Sechrist, Cory Shields, Wade Narrow, Tracy Withers, Crissy Dudgeon, Mike O'Dell, Aram Hezel, Darren Carpizo. The axon guidance receptor UNC5B is a driver of pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B021.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is its proclivity for metastasis as evidenced by the fact that 85% are stage IV at diagnosis. This highlights the need to better understand the biology of metastatic PDAC and identify novel therapies for this patient population. Axon guidance genes have been shown to be involved in PDAC progression, but their role is unclear. We have investigated the role of the axon guidance molecule Netrin-1 and its receptors Unc5b and DCC in PDAC. We found that in both murine and human samples that NTN1 expression is increased in metastatic PDAC and the quasi-mesenchymal subtype. Murine and TCGA data indicate that Unc5b is the dominant NTN1 receptor and genetic knock-down (KD) or knock-out (KO) of either Netrin-1 or Unc5b decreases migration, invasion, and cell survival in vitro and hepatic metastatic growth in vivo. The mechanism of Netrin-1 upregulation in metastatic PDAC is unknown. We found that hepatic stellate cell (HSC) secreted retinoic acid upregulates NTN1 through both an RXR/RAR and Elf mediated mechanism. To determine if NTN1 is involved in the process of HSC activation we found that recombinant NTN1 added to HSCs in vitro induced activation. We examined the livers of mice harboring orthotopic PDAC tumors using murine pancreatic cancer lines that were either NTN1 wild type (WT) or KO, and found that the NTN-expressing lines increased HSC activation providing evidence that NTN1 is important for long distance intercellular communication between primary pancreatic tumors and the pre-metastatic liver. We detected NTN1 within extracellular vesicles, and mice pre-conditioned with EVs from NTN1 KO cells demonstrated a decreased metastatic burden as compared mice preconditioned with NTN1 WT cells. Treatment of several murine PDAC models (autochthonous and metastatic) with a monoclonal antibody to NTN1 led to decreased metastases and increased survival. These studies reveal that NTN1 is upregulated in metastatic PDAC mediated by a novel mechanism that involves EVs, HSC activation and RXR/RAR signaling. These studies provide pre-clinical evidence to support a human clinical trial of anti-NTN1 therapy in PDAC. Citation Format: Crissy Dudgeon, Anthony Casabianca, Chris Harris, Igor Astsaturov, Charline Ogier, Xiaoyang Su, Jason Pitarresi, Wade Narrow, Fady Soliman, Tracy Withers, Patrick Mehlen, Darren Carpizo. Retinoic acid produced by hepatic stellate cells facilitates Netrin-1 mediated pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B020.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies with a 5-year survival rate for all stages of only 6%. While surgery remains the most effective treatment for pancreatic cancer, the majority of patients that undergo resection go on to die of pancreatic cancer recurrence. Pancreatic cancer undergoes early stage metastasis; wherein tumorigenic cells disseminate prior to the formation of invasive PDAC during the PanIN stage. The cellular and molecular mechanisms governing the processes of pancreatic cancer metastasis and recurrence following resection are poorly understood in part because there are no animal models of pancreatic cancer recurrence. To address this, we have adapted the Syngraft model, an orthotopic syngeneic mouse model using a pancreatic tumor cell line (Ink4a.1 luc/mcherry) derived from the genetically engineered conditional model (KrasG12D/+; Ink4a flox/flox) that has been modified with lentiviral vectors expressing mCherry and luciferase. Immunoproficient FVB mice were orthotopically injected with 100 Ink4a.1 cells and primary tumor growth was monitored by luciferase activity using the IVIS Spectrum. Mice underwent distal pancreatectomy with spleenectomy when the primary pancreatic tumor measured ∼107 photons/sec/cm2, with a mean time to surgical resection at 19 days. Metastatic recurrence to the liver (65%) was followed using the IVIS Spectrum, with a time to metastatic recurrence of 22 +/- 13 days. After 17 pancreatectomies, we observed the following recurrence patterns: 1) liver only (2/17), 2) liver and peritoneal (9/17), and 3) no recurrence (6/17) with a median follow time of 436 days. To investigate the molecular mechanisms of recurrence, we generated four metastatic cell lines from intrahepatic macrometastases. These metastatic cell lines and the control parental Ink4a.1 cell line were subjected to poly A RNA sequencing. Following data analysis by Tophat and Cufflinks, up and down regulation was determined by a 2-fold change in expression. We identified a total of 56 downregulated and 94 upregulated genes. Basp1, Vgll3, Cldn1, Gjb3, Gas6, Itm2a, and Tle3, were found to be downregulated while 26 genes, including Add2, Col6a3, Dpysl3, Ntn1, Selp, and Serpinf1, were upregulated in all four metastatic cell lines. Validation by Western blotting confirmed the upregulation of Netrin1 (Ntn1) as a strong candidate for the regulation of metastasis. Because the recurrence patterns following pancreatectomy recapitulate the human pancreatic cancer, we conclude the Syngraft model can be successfully adapted to study pancreatic cancer recurrence. Future work in the lab will characterize the function(s) of Netrin1 that contribute to the processes of pancreatic cancer metastasis. Citation Format: Crissy Dudgeon, Jeffrey Rosenfeld, Matthew Habel, Eric Collisson, Darren Carpizo. The expression landscape of pancreatic cancer recurrence following resection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-351.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR13) of the Conference Proceedings. Citation Format: Crissy Dudgeon, Jeffrey Rosenfeld, Eric Collisson, Darren Carpizo. The expression landscape of pancreatic cancer recurrence following resection. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B14.