Smoot, Rory L.

Background and Aims Existing therapeutic approaches to treat cholangiocarcinoma (CCA) have limited effectiveness, prompting further study to develop therapies for CCA. We report a mechanistic role for the heparan sulfate editing enzyme sulfatase 2 (SULF2) in CCA pathogenesis. Approach and Results In silico analysis revealed elevated SULF2 expression in human CCA samples, occurring partly through gain of SULF2 copy number. We examined the effects of knockdown or overexpression of SULF2 on tumor growth, chemoresistance, and signaling pathway activity in human CCA cell lines in vitro. Up‐regulation of SULF2 in CCA leads to increased platelet‐derived growth factor receptor beta (PDGFRβ)–Yes‐associated protein (YAP) signaling activity, promoting tumor growth and chemotherapy resistance. To explore the utility of targeting SULF2 in the tumor microenvironment for CCA treatment, we tested an anti‐SULF2 mouse monoclonal antibody, 5D5, in a mouse CCA xenograft model. Targeting SULF2 by monoclonal antibody 5D5 inhibited PDGFRβ–YAP signaling and tumor growth in the mouse xenograft model. Conclusions These results suggest that SULF2 monoclonal antibody 5D5 or related agents may be potentially promising therapeutic agents in CCA.

Abstract Background Aspartate aminotransferase/platelet ratio index (APRI) and albumin–bilirubin grade (ALBI) are validated prognostic indices implicated as predictors of postoperative liver dysfunction after hepatic resection. The aim of this study was to evaluate the relevance of the combined APRI/ALBI score for postoperative clinically meaningful outcomes. Methods Patients undergoing hepatectomy were included from the American College of Surgeons National Surgical Quality Improvement Program database. The association between APRI/ALBI score and postoperative grade C liver dysfunction, liver dysfunction-associated and overall 30-day mortality was assessed. Results A total of 12 055 patients undergoing hepatic resection from 2014 to 2017 with preoperative blood values and detailed 30-day postoperative outcomes were included (exploration cohort: January 2014 to December 2016; validation cohort: 2017). In the exploration cohort (8538 patients), the combination of both scores (APRI/ALBI) was significantly associated with postoperative grade C liver dysfunction, 30-day mortality, and liver dysfunction-associated 30-day mortality, and was superior to either score alone. The association with postoperative 30-day mortality was confirmed in multivariable analysis. A predictive model was generated using the exploration cohort. The predicted incidence of events closely followed the observed incidence in the validation cohort (3517 patients). Subgroup analyses of tumour types were used to generate disease-specific risk models to assess risk in different clinical scenarios. These findings informed development of a smartphone application (https://tellaprialbi.37binary.com). Conclusion The predictive potential of the combined APRI/ALBI score for clinically relevant outcomes such as mortality was demonstrated. An evidence-based smartphone application will allow clinical translation and facilitation of risk assessment before hepatic resection using routine laboratory parameters.

Background and Aims Platelet‐stored serotonin critically affects liver regeneration in mice and humans. Selective serotonin reuptake inhibitors (SSRIs) and serotonin noradrenalin reuptake inhibitors (SNRIs) reduce intraplatelet serotonin. As SSRIs/SNRIs are now one of the most commonly prescribed drugs in the United States and Europe and given serotonin’s impact on liver regeneration, we evaluated whether perioperative use of SSRIs/SNRIs affects outcome after hepatic resection. Approach and Results Consecutive patients undergoing hepatic resection (n = 754) were retrospectively included from prospectively maintained databases from two European institutions. Further, an independent cohort of 495 patients from the United States was assessed to validate our exploratory findings. Perioperative intake of SSRIs/SNRIs was recorded, and patients were followed up for postoperative liver dysfunction (LD), morbidity, and mortality. Perioperative intraplatelet serotonin levels were significantly decreased in patients receiving SSRI/SNRI treatment. Patients treated with SSRIs/SNRIs showed a higher incidence of morbidity, severe morbidity, LD, and LD requiring intervention. Associations were confirmed in the independent validation cohort. Combined cohorts documented a significant increase in deleterious postoperative outcome (morbidity odds ratio [OR], 1.56; 95% confidence interval [CI], 1.07‐2.31; severe morbidity OR, 1.86; 95% CI, 1.22‐2.79; LD OR, 1.96; 95% CI, 1.23‐3.06; LD requiring intervention OR, 2.22; 95% CI, 1.03‐4.36). Further, multivariable analysis confirmed the independent association of SSRIs/SNRIs with postoperative LD, which was closely associated with postoperative 90‐day mortality and 1‐year overall survival. Conclusions We observed a significant association of perioperative SSRI/SNRI intake with adverse postoperative outcome after hepatic resection. This indicates that SSRIs/SNRIs should be avoided perioperatively in patients undergoing hepatic resections.

Abstract Introduction: The effector protein of the Hippo pathway, Yes-associated protein (YAP), functions as a transcriptional coactivator and has been implicated in the biology of cholangiocarcinoma (CCA). Although the Hippo cascade consists of serine kinases, which act to sequester YAP in the cytosol, we have recently demonstrated the importance of tyrosine phosphorylation in regulating YAP localization and function in CCA. Furthermore, previous evaluation of clinical samples of CCA demonstrated a high frequency of mutations in tyrosine phosphatases. Evaluation of YAP-interacting proteins identified several tyrosine phosphatases, including SHP2. Herein, we describe the ability to modulate YAPY357 phosphorylation status, localization, and activity through manipulation of SHP2 levels and activity. Methods: The cholangiocarcinoma cell lines HuCCT-1 and KMCH were utilized. SHP2 activity was modulated utilizing the inhibitors NSC87877 and SHP099 (Selleckchem). SHP2 levels were modulated utilizing knockout via a doxycycline-inducible CRISPR/Cas9 system. Enforced expression of SHP2 was explored utilizing catalytically active and dead constructs. Tyrosine phosphorylation was assessed via immunoblot. Transcriptional activity was assessed by qRT-PCR for YAP target genes. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay utilizing SHP2 inhibitors and the Src-family kinase inhibitor dasatinib (Selleckchem). Results: The HuCCT-1 and KMCH cell lines were noted to have unique p-YAPY357 profiles (HuCCT-1 [p-YAPY357 high] KMCH [p-YAPY357 low]), and these profiles correlated with SHP2 levels and YAP subcellular localization with HuCCT1 cells demonstrating low levels of SHP2 and prominent nuclear YAP localization, while KMCH cells demonstrated high levels of SHP2 and predominately cytoplasmic YAP. In both cell lines, pharmacologic inhibition of SHP2 was accompanied by an increase in p-YAPY357 levels and YAP cotranscriptional activity (as noted by increased expression of cognate YAP target genes). In KMCH cells SHP2 inhibition shifted YAP from the cytoplasm to the nucleus, and these findings were confirmed by CRISPR/Cas9-mediated deletion of SHP2. In contrast, enforced expression of catalytically active SHP2 in HuCCT1 cells was associated with a decrease in p-YAPY357 levels and a shift of YAP from the nucleus to the cytoplasm. As predicted, based on an increase in YAP activity and nuclear localization, SHP2 inhibition was associated with an increase in cellular proliferation in both cell lines. Incubation of the cell lines with dasatinib was associated with decreased proliferation, which was partially rescued by pretreatment with an SHP2 inhibitor. Conclusions: The tyrosine phosphatase SHP2 can regulate YAPY357 phosphorylation, transcriptional coactivity, and subcellular localization in CCA. Inhibition of SHP2 activity protects cells from the effects of Src kinase inhibition on cellular proliferation and caspase activation. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Kabashima, Gregory Gores, Rory Smoot. The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A17.

Abstract The Hippo pathway effector Yes-associated protein (YAP) is localized to the nucleus and transcriptionally active in a number of tumor types, including a majority of human cholangiocarcinomas. YAP activity has been linked to chemotherapy resistance and has been shown to rescue KRAS and BRAF inhibition in RAS/RAF-driven cancers; however, the underlying mechanisms of YAP-mediated chemoresistance have yet to be elucidated. Herein, we report that the tyrosine phosphatase SHP2 directly regulates the activity of YAP by dephosphorylating pYAPY357 even in the setting of RAS/RAF mutations, and that diminished SHP2 phosphatase activity is associated with chemoresistance in cholangiocarcinomas. A screen for YAP-interacting tyrosine phosphatases identified SHP2, and characterization of cholangiocarcinomas cell lines demonstrated an inverse relationship between SHP2 levels and pYAPY357. Human sequencing data demonstrated lower SHP2 levels in cholangiocarcinomas tumors as compared with normal liver. Cell lines with low SHP2 expression and higher levels of pYAPY357 were resistant to gemcitabine and cisplatin. In cholangiocarcinomas cells with high levels of SHP2, pharmacologic inhibition or genetic deletion of SHP2 increased YAPY357 phosphorylation and expression of YAP target genes, including the antiapoptotic regulator MCL1, imparting resistance to gemcitabine and cisplatin. In vivo evaluation of chemotherapy sensitivity demonstrated significant resistance in xenografts with genetic deletion of SHP2, which could be overcome by utilizing an MCL1 inhibitor. Implications: These findings demonstrate a role for SHP2 in regulating YAP activity and chemosensitivity, and suggest that decreased phosphatase activity may be a mechanism of chemoresistance in cholangiocarcinoma via a MCL1-mediated mechanism.

Abstract Objectives Mucinous cystic neoplasm of the liver is characterized by neoplastic mucinous and/or biliary epithelium surrounded by ovarian-type stroma. Immunohistochemical studies have shown that the ovarian-type stroma expresses estrogen receptor, suggesting potential hormonal responsiveness. The molecular biology of mucinous cystic neoplasm of the liver remains poorly studied. Methods Transcriptome sequencing and immunohistochemistry were performed on a series of mucinous cystic neoplasms. Results Mucinous cystic neoplasm of the liver exhibited significantly increased RNA expression of ovarian stromal markers WT1, PR, and ER2 and sex cord stromal markers SF-1, inhibin-α, and calretinin compared with nonneoplastic liver. Immunohistochemistry confirmed the RNA-level data. Evidence for sex hormone biosynthesis was identified by significant overexpression of multiple estrogen biosynthetic enzymes. Expression of 17β-hydroxysteroid dehydrogenase 1 was confirmed immunohistochemically. Pathway analysis also identified significant upregulation of the hedgehog and Wnt pathways and significant downregulation of T-helper 1 and T-helper 2 pathways. Conclusions Mucinous cystic neoplasm of the liver recapitulates ovarian stroma at the morphologic, DNA, RNA, and protein levels. These data support the concept that this tumor likely arises from ectopic primitive gonadal tissue and/or stromal cells with capacity to transdifferentiate to ovarian cortical cells.

147 Background: Colorectal cancer is one of the leading causes of cancer related deaths with liver being most common site of CRC metastasis. More than 50% of the CRC patients will develop metastatic liver lesion that eventually leads to death in about 70% of them. In this retrospective review we reviewed the outcome of pts who received neoadjuvant chemotherapy followed by resection of liver lesion for metastatic colorectal cancer. Methods: 304 pts who had preoperative chemotherapy were identified from 1045 metastatic colorectal cancer patients who had liver metastasectomy at Mayo Clinic between 1997 and 2018. A retrospective review was conducted by using data from electronic medical records. Statistical analyses utilized Kaplan-Meier method, Log-rank test, and Cox proportional hazards models. Results: There were 113 (37%) female and 191 (63%) male pts. Median age at primary disease diagnosis was 56.5 yrs. Two hundred forty-nine pts presented with stage IV metastatic colorectal cancer. Primary tumor locations were: 53 right-sided, 117 left-sided and 133 rectum. 152 (50%) pts had extrahepatic metastases. Two pts were found to be MSI-H, 113 MSS, 189 unknown. BRAF mutation was found in 6 patients. RAS mutation was present in 84 patients, with 124 unknown. Pts received chemotherapy for median of 2.82 months. Single agent fluoropyrimidine was administered in 38 (12%) pts and rest receiving chemotherapy doublet or triplet with fluropyrimidine plus oxaliplatin being most common regimen. The median overall survival from primary diagnosis for the entire group was 74.5 months. Median overall survival from liver metastasectomy was 60.0 months. In univariate analysis, metachronous disease, age < 60 yrs, and an absence of extrahepatic lesions led to statistically significant improvement of overall survival from primary diagnosis. Metachronous and extrahepatic lesions remained statistically significant in multivariate analysis. Conclusions: Neoadjuvant chemotherapy followed by liver metastasectomy is beneficial for highly-selected metastatic colorectal cancer pts. Compared to a historical control of 30-36 months, our patient population had a median overall survival of about 5 years from resection.

AbstractBackground and ObjectivesConclusive evidence in favor of neoadjuvant therapy for those with non‐metastatic pancreatic ductal adenocarcinoma (PDAC) is still lacking. The objective of this study was to evaluate the survival benefit of neoadjuvant therapy vs upfront surgery for patients with non‐metastatic PDAC.MethodsThe study involved 565 patients undergoing neoadjuvant therapy or upfront surgery as the primary treatment for PDAC. Propensity score matching was performed between the neoadjuvant therapy group (NAT group) and the upfront surgery group (UFS group) using 20 clinical variables at diagnosis. Overall survival and surgical pathology were compared between the two treatment groups on an intent‐to‐treat basis.ResultsIn the matched cohort, the NAT group (n = 91) had a longer median overall survival than the UFS group (n = 91) (23.1 months vs 18.5 months, P = .043). The rate of patients undergoing surgical resection was lower in the NAT group (58% vs 80%, P = .001). Regarding surgical pathology, the NAT group had smaller tumor size (2.8 cm vs 4.0 cm, P = .001), lower incidence of positive surgical margins (8% vs 30%, P < .002), and less lymph node metastasis (45% vs 78%, P < .001).ConclusionsThe strategy of neoadjuvant therapy before surgical resection appears to offer pathologic effect and survival benefit for the patients presenting with non‐metastatic PDAC.

Abstract Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation. Methods: In silico analysis of the TCGA cholangiocarcinoma dataset was undertaken specifically evaluating tyrosine phosphatase levels. Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH as well as the murine CCA cell line SB1 (expressing a flag-tagged YAP). Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. Tyrosine phosphorylation was inhibited utilizing the multi-kinase inhibitor dasatinib. The pan-tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) and the selective SHP1/2 inhibitor NSC87877 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR. Apoptosis was evaluated by caspase 3/7 assay. Results: Multiple tyrosine phosphatases were noted to be expressed at lower levels in tumor versus normal adjacent liver in the TCGA RNAseq dataset. YAP-interacting tyrosine phosphatases identified by co-IP included PTPN1, 11, 23, and PTPRK. Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. Conversely, incubation of HuCCT-1 cells with dasatinib rapidly decreased p-YAPY357 levels; and this effect was diminished by pre-incubation with either Na3VO4or NSC87877. The effect on YAP tyrosine phosphorylation paralleled effects on apoptosis; incubation of HuCCT-1 cells with dasatinib lead to an apoptotic response as measured by caspase 3/7 assay, which was eliminated by pre-incubation with Na3VO4 or NSC87877. Conclusions: Inhibition of the tyrosine phosphatase SHP2 is associated with elevated p-YAPY357 levels, elevated YAP co-transcriptional activity, and a blunted therapeutic response to dasatinib in cholangiocarcinoma cell lines. Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Niibe, Gregory Gores, Rory Smoot. Tyrosine phosphatase activity is required for response to Src kinase inhibition in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3449.