ROR2 regulates cellular plasticity in pancreatic neoplasia and adenocarcinoma.

Cellular plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) starting from the conversion of normal cells into precancerous lesions to the progression of carcinoma subtypes associated with aggressiveness and therapeutic response. We discovered that normal acinar cell differentiation, maintained by the transcription factor Pdx1, suppresses a broad gastric cell identity that is maintained in metaplasia, neoplasia, and the classical subtype of PDAC in mouse and human. We have identified the receptor tyrosine kinase Ror2 as marker of a gastric metaplasia (SPEM)-like identity in the pancreas. Ablation of Ror2 in a mouse model of pancreatic tumorigenesis promoted a switch to a gastric pit cell identity that largely persisted through progression to the classical subtype of PDAC. In both human and mouse pancreatic cancer, ROR2 activity continued to antagonize the gastric pit cell identity, strongly promoting an epithelial to mesenchymal transition, conferring resistance to KRAS inhibition, and vulnerability to AKT inhibition.

ROR2 Regulates Cellular Plasticity in Pancreatic Neoplasia and Adenocarcinoma.

Cellular plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) starting from the conversion of normal cells into precancerous lesions, to the progression of carcinoma subtypes associated with aggressiveness and therapeutic response. We discovered that normal acinar cell differentiation, maintained by the transcription factor Pdx1, suppresses a broad gastric cell identity that is maintained in metaplasia, neoplasia, and the classical subtype of PDAC in mouse and human. We have identified the receptor tyrosine kinase Ror2 as marker of a gastric metaplasia-like identity in pancreas neoplasms. Ablation of Ror2 in a mouse model of pancreatic tumorigenesis promoted a switch to a gastric pit cell identity that largely persisted through progression to the classical subtype of PDAC. In both human and mouse pancreatic cancer, ROR2 activity continued to antagonize the gastric pit cell identity, strongly promoting an epithelial to mesenchymal transition, conferring resistance to KRAS inhibition, and vulnerability to AKT inhibition.

Lorazepam Stimulates IL6 Production and Is Associated with Poor Survival Outcomes in Pancreatic Cancer.

PURPOSE: This research investigates the association between benzodiazepines (BZD) and cancer patient survival outcomes, the pancreatic cancer tumor microenvironment, and cancer-associated fibroblast (CAF) signaling. EXPERIMENTAL DESIGN: Multivariate Cox regression modeling was used to retrospectively measure associations between Roswell Park cancer patient survival outcomes and BZD prescription records. IHC, H&E, Masson's trichrome, RNAscope, and RNA sequencing were used to evaluate the impact of lorazepam (LOR) on the murine PDAC tumor microenvironment. ELISA and qPCR were used to determine the impact of BZDs on IL6 expression or secretion by human-immortalized pancreatic CAFs. PRESTO-Tango assays, reanalysis of PDAC single-cell sequencing/TCGA data sets, and GPR68 CRISPRi knockdown CAFs were used to determine the impact of BZDs on GPR68 signaling. RESULTS: LOR is associated with worse progression-free survival (PFS), whereas alprazolam (ALP) is associated with improved PFS, in pancreatic cancer patients receiving chemotherapy. LOR promotes desmoplasia (fibrosis and extracellular matrix protein deposition), inflammatory signaling, and ischemic necrosis. GPR68 is preferentially expressed on human PDAC CAFs, and n-unsubstituted BZDs, such as LOR, significantly increase IL6 expression and secretion in CAFs in a pH and GPR68-dependent manner. Conversely, ALP and other GPR68 n-substituted BZDs decrease IL6 in human CAFs in a pH and GPR68-independent manner. Across many cancer types, LOR is associated with worse survival outcomes relative to ALP and patients not receiving BZDs. CONCLUSIONS: We demonstrate that LOR stimulates fibrosis and inflammatory signaling, promotes desmoplasia and ischemic necrosis, and is associated with decreased pancreatic cancer patient survival.

Targeting the oncogenic protein beta-catenin to enhance chemotherapy outcome against solid human cancers.

BACKGROUND: Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of ß-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3ß) through phosphorylation. Cell growth and proliferation is associated with ß-catenin translocation from the cytoplasm into the nucleus. This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on ß-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of ß-catenin, (2) investigate the kinetics and the mechanism of ß-catenin inhibition, and (3) confirm that inhibition of ß-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs. RESULTS: In six human cancer cell lines, the inhibition of total and nuclear expression of ß-catenin by MSeA was dose and time dependent. The involvement of GSK-3ß in the degradation of ß-catenin was cell type dependent (GSK-3ß-dependent in HT-29, whereas GSK-3ß-independent in HCT-8). However, the pronounced inhibition of ß-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.Knockout of ß-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.Collectively, the generated data demonstrate that ß-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs. CONCLUSIONS: This study demonstrates that ß-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of ß-catenin by GSK-3ß is not a general mechanism but is cell type dependent.

Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons.

Resistance of human renal cell carcinoma (RCC) and melanoma to the apoptosis-inducing effects of IFNs was postulated to result from epigenetic silencing of genes by DNA methylation, a common feature of human cancers. To reverse silencing, 5-AZA-deoxycytidine (5-AZA-dC) or selective depletion of DNA methyltransferase 1 (DNMT1) by phosphorothioate oligonucleotide antisense (DNMT1 AS) were employed in cells resistant (<5% terminal deoxynucleotidyl transferase-mediated nick-end labeling positive) to apoptosis induction by IFN-alpha2 and IFN-beta (ACHN, SK-RC-45, and A375). 5-AZA-dC and DNMT1 AS similarly depleted available DNMT1 protein and, at doses that did not cause apoptosis alone, resulted in apoptotic response to IFNs. The proapoptotic tumor suppressor RASSF1A was reactivated by DNMT1 inhibitors in all three cell lines. This was associated with demethylation of its promoter region. IFNs augmented RASSF1A protein expression after reactivation by DNMT1 inhibition. In IFN-sensitive WM9 melanoma cells, expression of RASSF1A was constitutive but also augmented by IFNs. RASSF1A small interfering RNA reduced IFN-induced apoptosis in WM9 cells and in DNMT1-depleted ACHN cells. Conversely, lentiviral expression of RASSF1A but not transduction with empty virus enabled IFN-induced apoptosis. IFN induced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL-neutralizing antibody inhibited apoptotic response to IFN in RASSF1A-expressing ACHN cells. Accordingly, RASSF1A markedly sensitized to recombinant TRAIL. Normal kidney epithelial cells, although expressing RASSF1A, did not undergo apoptosis in response to IFN or TRAIL but had >400-fold higher TRAIL decoy receptor 1 expression than transduced ACHN cells (real-time reverse transcription-PCR). Results identified RASSF1A as regulated by IFNs and participating in IFN-induced apoptosis at least in part by sensitization to TRAIL.

The prolyl isomerase Pin1 targets stem-loop binding protein (SLBP) to dissociate the SLBP-histone mRNA complex linking histone mRNA decay with SLBP ubiquitination.

Histone mRNAs are rapidly degraded at the end of S phase, and a 26-nucleotide stem-loop in the 3' untranslated region is a key determinant of histone mRNA stability. This sequence is the binding site for stem-loop binding protein (SLBP), which helps to recruit components of the RNA degradation machinery to the histone mRNA 3' end. SLBP is the only protein whose expression is cell cycle regulated during S phase and whose degradation is temporally correlated with histone mRNA degradation. Here we report that chemical inhibition of the prolyl isomerase Pin1 or downregulation of Pin1 by small interfering RNA (siRNA) increases the mRNA stability of all five core histone mRNAs and the stability of SLBP. Pin1 regulates SLBP polyubiquitination via the Ser20/Ser23 phosphodegron in the N terminus. siRNA knockdown of Pin1 results in accumulation of SLBP in the nucleus. We show that Pin1 can act along with protein phosphatase 2A (PP2A) in vitro to dephosphorylate a phosphothreonine in a conserved TPNK sequence in the SLBP RNA binding domain, thereby dissociating SLBP from the histone mRNA hairpin. Our data suggest that Pin1 and PP2A act to coordinate the degradation of SLBP by the ubiquitin proteasome system and the exosome-mediated degradation of the histone mRNA by regulating complex dissociation.