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Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5− state in response to chemotherapy, LGR5− targeted antibody–drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody–drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5− colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.

Antibody-drug conjugates (ADCs) have become an increasingly successful class of anticancer therapy, particularly within the past few years. Though ADCs are in clinical trials for colorectal cancer (CRC), a candidate has yet to be approved. CRC continues to be a leading cause of cancer-related death, emphasizing the need to identify novel target antigens for ADC development. GPR56, a member of the 7TM receptor family, is upregulated in colorectal tumors compared to normal tissues and located on the surface of CRC cells, making it a promising ADC target. Furthermore, high GPR56 expression occurs in tumors that are microsatellite stable, negative for the CpG methylator phenotype, and show chromosomal instability. We previously reported the generation of a high affinity GPR56-specific monoclonal antibody, 10C7, and we have now mapped the epitope to the C-terminal end of the extracellular domain, proximal to the GPCR proteolysis site. Here, we describe the development of a duocarmycin-conjugated 10C7 ADC. 10C7 co-internalized with GPR56 and trafficked to the lysosomes of CRC cells, which is critical for efficient ADC payload release. Evaluation of the ADC in a panel of CRC cell lines and tumor organoids with different levels of GPR56 expression showed the ADC selectively induced cytotoxicity at low nanomolar concentrations in a GPR56-dependent manner. A nontargeting control ADC showed minimal to no activity. Furthermore, GPR56 ADC exhibited significant antitumor efficacy against GPR56-expressing patient-derived xenograft models of CRC. This study provides rationale for the development of a GPR56-targeted ADC approach to potentially treat a large fraction of CRC patients.

Cancer stem cell (CSC) marker LGR5 is highly expressed in colorectal cancer (CRC) and has been considered as a therapeutic target for the treatment of CRC. Previously, we and others showed that CRC tumors were eliminated by treatment with LGR5-targeted antibody-drug conjugates (ADCs). However, a fraction of tumors eventually recurred, likely due to CSC plasticity or the ability of LGR5-positive CSCs to interconvert into more drug resistant LGR5-negative cancer cells. Through our efforts to identify underlying mechanisms of drug resistance and plasticity and develop therapies to eliminate CRC, we showed that GPR56 expression is increased with loss of LGR5 protein expression. GPR56 is a member of the adhesion G-protein coupled receptor (GPCR) family which mediates diverse functions, including cell adhesion and tumorigenesis. GPR56 is also upregulated in CRC and increased expression levels correlate with poor patient survival. We recently showed GPR56 can promote tumor growth and drug resistance of CRC cells through upregulation of multidrug resistance proteins. These findings identify GPR56 as a potential therapeutic target for the treatment of CRC. Presently, we are focused on the development and characterization of anti-GPR56 ADCs that selectively bind GPR56-high CRC cells to deliver potent cytotoxic drugs and destroy them. We reported the generation of unique anti-GPR56 monoclonal antibodies (mAbs) and are currently developing and optimizing GPR56-targeted ADCs conjugated with different drugs. Cell-based binding assays show that our anti-GPR56 mAb binds with high affinity and specificity to 293T cells overexpressing recombinant GPR56 or GPR56-high CRC cells, with no detectable binding to GPR56-negative cells. Using cytotoxicity assays, we evaluated the in vitro efficacy of anti-GPR56 ADCs against several CRC lines with different levels of GPR56 expression and showed that the ADC is only effective against CRC cells expressing GPR56. Moreover, we evaluated the therapeutic efficacy of our anti-GPR56-ADC against CRC cell line and patient-derived tumor xenograft models in vivo and demonstrated significant inhibition of tumor growth. Altogether, these findings suggest that novel GPR56-targeted ADCs may be a promising therapeutic strategy to overcome resistance for the improved treatment of CRC. Citation Format: Liezl E. Francisco, Treena Chatterjee, Zhengdong Liang, Sheng Zhang, Kendra Carmon. GPR56 as a therapeutic target for the development of antibody-drug conjugates for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6328.

Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. This is largely due to metastasis, tumor relapse, and the vast heterogeneity of CRC tumors leading to treatment failure. Cancer stem cells (CSCs) are a small population of tumor-initiating cells thought to act as immortal seeds that can form metastases and contribute to tumor relapse. CSCs have been shown to exhibit plasticity or the ability to transition between differentiated and undifferentiated states to evade treatment and to promote tumor progression. Epiregulin (EREG) is an EGFR family ligand that is highly expressed in both states, as well as in treatment resistant CRCs of various mutation statuses. We showed that knock-out of EREG in a CRC cell line resulted in significant tumor inhibition in vivo, suggesting EREG is a promising target for therapeutic development. Therefore, we generated EREG-targeted antibody-drug conjugates (ADCs) to act as guided missiles for the delivery of cytotoxic drugs into EREG-expressing tumors to overcome plasticity and resistance in CRC. To develop a novel EREG ADC, we cloned and produced three separate EREG monoclonal antibody (mAbs) and tested them for binding, the ability to internalize into CRC cells, and to neutralize EREG activity. All three EREG mAbs were shown to bind with high affinity to EREG tethered to the surface of CRC cells and internalize to the lysosome, which is important for processing the release of drug from the ADC. We then conjugated the highest affinity EREG mAb and a non-targeting isotype control antibody to a highly cytotoxic drug via a cleavable peptide linker. EREG ADCs were evaluated in vitro against a wide panel of CRC cell lines of various Kras, Braf, and PI3CKA mutational statuses and different levels of EREG expression. Drug efficacy was determined using cytotoxicity assays to measure changes in CRC cell survival with escalating doses of unconjugated control antibody and EREG mAb, as well as control ADC (cADC) and EREG ADC. EREG mAb alone did not result in significant CRC cell death. However, EREG ADCs exhibited potent cell-killing in EREG-expressing CRC cells, whereas minimal effects were observed with cADC. Future progress will involve testing the safety and efficacy of EREG ADCs against patient-derived tumor xenograft models of CRC in vivo. These preliminary results demonstrate the therapeutic potential of EREG ADCs for targeting plasticity and drug resistance in CRC. Citation Format: Joan Jacob, Zhengdong Liang, Kendra Carmon. Development and characterization of an epiregulin antibody-drug conjugate for targeting colorectal cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4251.

e16290 Background: The mechanism of human pancreatic ductal adenocarcinoma (PDAC) resistance to nucleoside analog and first-line chemotherapy drug gemcitabine is not clearly understood, but some studies have associated it with increased autophagy. In cancer biology, autophagy plays dual roles as it can promote tumor suppression during early stages, but in established tumors, it plays a crucial role in tumor growth by enhancing survival under metabolic and therapeutic stress. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of a network of tumorigenic factors, including the Valosin-containing protein (VCP), which has been reported to play an important role in autophagy. In this study, we investigate whether the repression of VCP through miR-198 replacement disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment. Methods: MIAPaCa2 cells with forced overexpression of mesothelin (MSLN) and AsPC-1 cell lines and, CDX and PDX mouse models were used for gemcitabine sensitization studies. For miR-198 replacement, a miR-198 expression vector-loaded lactic co-glycolic-acid-modified polyethylenimine polyplex (LPNP-p198) was used. Autophagy disruption experiments were run over miR-198 and/or VCP overexpressing cell lines. Nude mice were used for subcutaneous and orthotopic CDX models and SCID/Beige were used for the subcutaneous PDX model. Results: Cell lines were treated with LPNP-p198 in combination with gemcitabine and cell growth was significantly inhibited when compared to gemcitabine alone. Additionally, it was determined that miR-198 disrupts the autophagy maturation process and, that it is then restored by overexpression of VCP. LPNP-p198 can effectively enter tumor cells and induce tumor sensitization in vivo, resulting in an 80-90% reduction of tumor burden and metastatic spread in the LPNP-p198 plus gemcitabine group when compared to controls, with the concomitant downregulation of VCP expression in the tumor tissue. In addition, this group had the fewest mice with jaundice, ascites, and metastases to the abdominal cavity, spleen, liver, and kidney. Finally, to address the potential of the observed effect in the context of the heterogenic nature of PDAC, we confirmed our findings in a PDX mouse model, where a marked reduction in tumor burden was observed in the LPNP-p198 plus gemcitabine group compared to controls. Conclusions: Our findings indicate that miR-198 replacement disrupts the autophagy maturation process and sensitizes PDAC cells to gemcitabine through VCP repression, indicating a potential therapeutic strategy for targeting gemcitabine-resistant PDAC and, establishing the use of LPNPs as a prototype for effective nucleic acid delivery in vitro and in vivo, with potential to be used from bench to clinic.

In humans, the NK cell marker CD161 identifies several subsets of T cells, including a polyclonal CD8 αβ T cell receptor expressing subset with characteristic specificity for tissue-localized viruses. This subset has also been reported to display enhanced cytotoxic and memory phenotypes. Here, we characterized the biology and functional properties of this unique T cell subset and determined its potential suitability for use in chimeric antigen receptor (CAR) T cell therapy. In mice, gene expression profiling among the CD161 equivalent CD8(+) T cell populations (CD8(+)NK1.1(+)) revealed substantial upregulation of granzymes, perforin, killer lectin-like receptors and innate signaling molecules in comparison to CD8(+)NK1.1(−) T cells. Adoptive transfer of CD8(+)NK1.1(+) cells from previously exposed animals offered substantially enhanced protection and improved survival against melanoma tumors and influenza infection compared to CD8(+)NK1.1(−) cells. Freshly isolated human CD8(+)CD61(+) T cells exhibited heightened allogeneic killing activity in comparison to CD8(+)CD61(−) T cells or total peripheral blood mononuclear cells (PBMC). To determine if this subset might be utilized to improve the antitumor efficacy CAR T cell therapy in the solid tumor setting, we compared bulk PBMC, CD8(+)CD161(−), and CD8(+)CD161(+) T cells transduced with a HER2-specific CAR construct. In vitro, CD8(+)CD161(+) CAR-transduced T cells killed HER2(+) targets faster and with greater efficiency. Similarly, these cells mediated substantially enhanced in vivo anti-tumor efficacy and survival in xenograft models of HER2(+) pancreatic ductal adenocarcinoma, exhibiting elevated expression of granzymes and reduced expression of exhaustion markers. These data suggest that this unique T cell subset presents an opportunity to improve CAR T cell therapy for the treatment of solid tumors.

Background: High mesothelin (MSLN) expression has been associated with poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC); however, the effect of MSLN exert on PDAC tumor microenvironment (TME) is largely unknown. Methods: MSLN over-expressed (MSLN-OE) and knocked-down (MSLN-KD) PDAC cells were established to investigate the MSLN-PD-L1 relationship in PDAC. Using an MSLN overexpressed Panc02 (Panc02-MSLN) cell line, we established an orthotopic PDAC mouse model. All mice were euthanized on day 27 after cell inoculation and the tumor-infiltrating leukocytes (TILs) were isolated for mass cytometry (CyTOF) study. The gene expression correlation between MSLN and a panel of immune cell markers was assessed by using a dataset that includes149 PDAC patients in the TCGA-PAAD cohort. Results: MSLN upregulated PD-L1 expression at both mRNA and protein levels in PDAC. MSLN was able to enhance PD-L1 transcription by recruitment of NF-kB P65 to the PD-L1 promoter. The Orthotopic implanted Panc02-MSLN group showed significantly increased tumor size, tumor weight, upregulated PD-L1, and reduced survival when compared with the vector control group. CyTOF analysis of the total CD45+ hematopoietic cells infiltrated to the TME revealed 13 distinct cell subsets. In MSLN-high tumors, there is a significant expansion of neutrophils, eosinophils, PD-1+CD8+, and CD8+NK cells. In addition, pan T cells, regulatory T cells (Treg), CD8+, and PD-1+CD4+ cells were increased. A considerable decrease of tumor-associated macrophages (TAM), dendritic cells, B cells, NK cells, and a trend towards decreased Ly6C+CD8+ cells was also found. Within the TAM subset, a decreased iNOS+M1 to Relm-a+M2 ratio was detected. Furthermore, although there were increased CD8+NK cells, we found more immunosuppressive markers such as PD-1, CTLA4, LAG3, KLRG1, and TIM3, and a decreased activation marker CD69 and transcription factor T-bet. Intriguingly, gene expression correlation analysis with 149 PDAC patients cohort in TCGA dataset showed that MSLN expression negatively correlates with the mRNA levels of the B220 marker of B cells, the F4/80 marker of macrophages, as well as the CD4, CD8, CD69, granzyme B, IL-2 and IFN-γ markers of T-cell activation. Conclusions: We showed here that MSLN could significantly modulate the TME, possibly through the upregulation of PD-L1 expression. Compared with MSLN-low PDAC tumors, MSLN-high tumors recruit diverse immunosuppressive rather than immunoreactive leukocytes into tumor tissues to constitute an immunologically cold TME. Since targeting MSLN or PD-L1 as a monotherapy exhibited only modest objective response rates in the clinic, our findings may provide an alternative strategy by combining the depletion of MSLN with immune checkpoint inhibitors, which could result in improved treatment efficacy in PDAC. Citation Format: Dongliang Liu, Ethan Poteet, Zhengdong Liang, Emily Laplante, Lisa Brubaker, Sadhna Dhingra, Aleksandar Milosavljevic, Changyi Chen, Qizhi Cathy Yao. Effects of mesothelin exert on tumor microenvironment in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-115.

Clinical trials of EGFR inhibitors in combination with gemcitabine for the treatment of pancreatic ductal adenocarcinoma (PDAC) have generated mixed results partially due to the poorly defined effectiveness of EGFR inhibitors in PDAC. Here, we studied a panel of PDAC cell lines to compare the IC50s of the EGFR inhibitors gefitinib and cetuximab. We found that gefitinib induced biphasic inhibition in over 50% of PDAC cells, with the initial growth inhibition occurring at nanomolar concentrations and a second growth inhibition occurring outside the clinical range. In contrast to gefitinib, cetuximab produced a single phase growth inhibition in a subset of PDAC cells. Using this sensitivity data, we screened for correlations between cell morphology proteins and EGFR ligands to EGFR inhibitor sensitivity, and found that mesothelin and the EGFR ligand TGF-α have a strong correlation to gefitinib and cetuximab sensitivity. Analysis of downstream signaling pathways indicated that plc-γ1 and c-myc were consistently inhibited by EGFR inhibitor treatment in sensitive cell lines. While an inconsistent additive effect was observed with either cetuximab or gefitinib in combination with gemcitabine, the cell pathway data indicated consistent ERK activation, leading us to pursue EGFR inhibitors in combination with trametinib, a MEK1/2 inhibitor. Both cetuximab and gefitinib in combination with trametinib produced an additive effect in all EGFR sensitive cell lines. Our results indicate that mesothelin and TGF-α can predict PDAC sensitivity to EGFR inhibitors and a combination of EGFR inhibitors with trametinib could be a novel effective treatment for PDAC.

Pancreatic cancer is the 12th most common cancer, but is the 3rd leading cause of cancer death in America. This disparity is directly related to three factors: the difficulty in early detection of the disease, pancreatic cancer’s resistance to chemotherapy, and a tendency for the cancer to metastasize early and quickly. Here, we propose a novel immunotherapy to bypass pancreatic cancer’s resistance to standard chemotherapy. We have developed a virus-like particle (VLP) based on an HIV backbone Gag and pancreatic cancer-associated protein mesothelin (MSLN). Vaccination of C57BL/6 mice with MSLN VLP can efficiently activate the immune system and reduce and in some cases eliminate the host’s pancreatic cancer. Our results show that MSLN VLPs induce both humoral and cytotoxic specific response against MSLN in mouse pancreatic cancer models. Specifically, after vaccination with MSLN VLPs, significant increases in CD8+ MSLN specific T-cells and increases in serum anti-MSLN IgG were detected. Furthermore, we observed an increase in the serum of pro-inflammatory cytokine IFN-γ and B cell stimulator IL-5. Consistent with the increased IL-5, we observed a significant increase in tumor infiltrating B cells in the MSLN VLP immunized groups, but neither Gag VLP control nor unimmunized mice. Furthermore, while all tumor-bearing mice generated some antibodies towards MSLN, our MSLN VLP immunized mice had significantly higher serum titers of IgG1, IgG2B, and IgG3. Both Gag VLP and MSLN VLP immunized mice had a decreased tumor volume compared to control, but passive transfer of sera only from MSLN VLP immunized mice decreased tumor volume and increased survival of unimmunized tumor bearing mice, while Gag VLP immunized mice had much less effect. Overall, we have developed a promising vaccine for pancreatic cancer warranting additional studies with adjuvants and combination treatments with standard-of-care chemotherapy. Citation Format: Qizhi Cathy Yao, Ethen Poteet, Zhengdong Liang, Phoebe Lewis, Zhiyin Yu, Changyi Chen. Anti-tumor immune responses elicited by mesothelin virus-like particles vaccine effectively controls pancreatic cancer in animal models [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B141.

Alterations in arachidonic acid metabolism are involved in human carcinogenesis. Cyclooxygenase (COX) and lipoxygenase (LOX) are key enzymes in this metabolism. We analyzed the expression of 15S-lipoxygenase-2 (15-LOX-2) mRNA and protein in surgical specimens from normal (N=37) and malignant (63) esophageal tissues using in situ hybridization and immunohistochemistry (IHC), and in normal (1), premalignant (1), and malignant (5) esophageal cell lines using Northern and Western blotting. 15-LOX-2 was expressed in normal esophageal epithelial cells (EECs) at the highest levels, whereas an SV40-immortalized HET-1A line and three of five esophageal cancer cell lines failed to express it at detectable levels. 15-LOX-2 was detected in 76% (28/37) of the normal esophageal mucosae, but only in 46% (29/63) of the cancer specimens using IHC (P

Background: Better understanding of the molecular mechanisms of pancreatic adenocarcinoma (PDAC) metastasis is important for the development of effective treatments for this devastating disease. We have previously shown that overexpression of Semaphorin 3E (Sema3E) enhances PDAC cell growth and is associated with poor patient survival. Here, we further determined the effect of Sema3E on stromal cells and the molecular mechanisms contributing to PDAC metastasis. Materials and Methods: Panels of stable PDAC cell lines were generated using either lenti-Sema3E for overexpression or the Crispr/Cas9 system to knock out (KO) Sema3E in both panc28 and panc48 cell lines. The effects of Sema3E overexpression or KO were measured in orthotopically implanted xenograft tumor mouse models in respect to tumor size, metastasis, and survival. After coculture with pancreatic stellate cells (PSCs), the effects of Sema3E on PSC activation were evaluated by GFAP and αSMA staining. Mixtures of PDAC cell lines with PSCs in vivo tumor growth were also evaluated. Result: After orthotopically implanting a panel of Sema3E-overexpressing or knockout panc28 cell lines, we found that the survival span of the sema3E-overexpression cell implant group was significantly shorter than the vector control group (31±9 days vs. 51±15 days) (p Citation Format: Zhengdong Liang, Lin-Kin Yong, George Van Buren, William Fisher, Rosa Hwang, Changyi Chen, Qizhi Cathy Yao. Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B031.

Combination therapy for pancreatic adenocarcinoma (PDAC) has recently had two successful stage III trials with FOLFIRINOX (combination folinic acid, fluorouracil, irinotecan, and oxalplatin) and combination gemcitabine and paclitaxel. Because of the heterogeneity of PDAC, the benefits of combination therapy are immediately apparent since it targets multiple facets of cancer simultaneously. However, FOLFIRINOX and gemcitabine + paclitaxel are highly toxic and affect all dividing cells, making apparent the need for better understanding of the cell profiles in PDAC. The epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many cancers, with EGFR or its ligands often overexpressed or mutated. Recently we have found that EGFR expression is correlated to mesothelin (MSLN) expression in PDAC. MSLN, a cell surface glycoprotein, has been shown by us and other labs to be overexpressed in 80 to 90% of all PDAC cases. To examine the relationship between MSLN and EGFR, we modulated MSLN expression by generating a MSLN overexpressing cell line from MIA-PaCa2 cells, and a MSLN knockdown cell line from CAPAN-2 cells. The change in EGFR signaling mimicked that of MSLN, with MSLN overexpression significantly increasing EGFR expression and MSLN knockdown significantly decreasing EGFR expression. Furthermore, high MSLN (and therefore high EGFR expression) showed increased sensitivity to gefitinib, an EGFR ATP binding site inhibitor, at concentrations as low as 100 nM, while MSLN low expressing cells did not exhibit any effect from gefitinib at concentrations below 10 µM. Cell Cycle analysis demonstrated that gefitinib increased the number of cells in G1 phase after 24 hours in MSLN high expressing cells, but had no effect on MSLN low expressing cells. In addition, western blot analysis of EGFR downstream pathways showed an increase in phosphorylated phospholipase C-Υ (PLC-Υ) in MSLN high expressing cells, which could be mitigated by treatment with gefitinib. Finally, combination therapy with gemcitabine and 100 nM gefitinib showed an additive effect in MSLN high expressing cells, while in MSLN low expressing cells combination gemcitabine and gefitinib had no additional effect over gemcitabine alone. This study demonstrates the correlation between MSLN expression and EGFR expression and indicates that gefitinib could be a potential addition to combination therapy for PDAC in patients with high MSLN expression. Citation Format: Ethan Poteet, Zhengdong Liang, Youwen Wang, Changyi Chen, Qizhi Yao.{Authors}. Mesothelin confers tumor cell vulnerabilities for gefitinib treatment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A90.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the US with a 5-year survival rate of less than 6%. There is an urgent need for a better understanding of PDAC in order to find better therapeutic targets. Two recent large-scale genomic analyses of human PDAC have uncovered increased copy numbers of an axon-guidance gene SEMA3E that codes for Semaphorin-3E (Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon cancers, particularly in promoting tumor cell migration and invasion, as well as inducing epithelial-to-mesenchymal transition (EMT). Here, we evaluated Sema3E expression levels in human pancreatic cancer tissue samples and correlation with patient survival; we further studied roles of Sema3E in pancreatic cancer cell proliferation. Materials and Methods: To determine expression levels of Sema3E in PDAC, qPCR analysis was performed on resected human PDAC samples and matched tissue controls, and immunohistochemical (IHC) analysis was performed on resected human PDAC tissues and tissues from a mouse model of PDAC. To generate Sema3E-overexpressing stable cells, human PDAC cell lines were transduced with Sema3E-expressing lentivirus or vector control. To generate Sema3E knock-down cell lines, CRISPR/Cas9 system was used to target Sema3E in the genome. MTT assay was performed on Sema3E-overexpressed cells to determine the effects of Sema3E on cell proliferation. Results: Using qPCR, Sema3E was found to be significantly upregulated in 46% of human PDAC samples (n=24), with an average fold-change of 9.54. IHC analyses of human and mouse PDAC revealed higher expression of Sema3E in tumor cells compared to normal cells (n=54). Interestingly, in human PDAC, Sema3E expression was concentrated in the nuclei of tumor cells while it has been reported to be a secreted protein. We found a positive correlation between high N/C ratio of Sema3E expression (i.e. expression in the nucleus relative to the cytoplasm) and poor survival. In addition, we also found overexpression of Sema3E is correlated with patient poor survival by analyzing a TCGA dataset (n=78). Overexpression of Sema3E in human PDAC cell lines enhanced cell proliferation and migration in vitro, while knocking-down Sema3E resulted in reduced cell proliferation. Conclusions: Our findings indicate that Sema3E is overexpressed in PDAC and is correlated with patient poor survival. Sema3E may play a pathogenic role in PDAC progression by enhancing tumor cell proliferation. These findings suggest that Sema3E could be an attractive novel therapeutic target for PDAC. Citation Format: Lin-Kin Yong, Syeling Lai, Zhengdong Liang, Dali Li, Ethan Poteet, William Fisher, Qianxin Mo, Changyi Chen, Qizhi Yao. Overexpression of Semaphorin-3E enhances pancreatic cancer cell proliferation ad is associated with patient poor survival. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A48.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the US with a 5-year survival rate of less than 6%. There is an urgent need for a better understanding of PDAC in order to find better therapeutic targets. Two recent large-scale genomic analyses of human PDAC have uncovered increased copy numbers of an axon-guidance gene SEMA3E that codes for Semaphorin-3E (Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon cancers, particularly in promoting tumor cell migration and invasion, as well as inducing epithelial-to-mesenchymal transition (EMT). We hypothesize a possible new role of an axon-guidance gene Sema3E in PDAC progression by promoting cell proliferation, invasion, and metastasis. Materials and Methods: To determine expression levels of Sema3E in human PDAC, qRT-PCR and IHC analysis was performed on resected PDAC samples and matched tissue controls. To determine Sema3E levels in mouse PDAC, IHC analysis was performed on pancreas tissue from a genetically engineered spontaneous PDAC mouse model, the PDX-1-Cre; KrasG12D; P53R172H (KPC) mouse. To generate Sema3E-overexpressing stable cells, human PDAC cell lines Mia PaCa-2 and Panc-48 cells were transduced with Sema3E-expressing lentivirus or vector control. QRT-PCR and western blot analyses were performed to verify the overexpression of Sema3E. Colony formation assay (CFA) and MTT assay were performed on Sema3E-transduced cells to determine the effects of Sema3E on cell survival and proliferation respectively, while a wound healing assay and boyden chamber assay were performed to determine effects of Sema3E on cell migration and invasion respectively. Results: Sema3E was found to be significantly upregulated in 46% of patient PDAC samples (n = 24), with an average fold-change of 9.54 across the samples. IHC analyses of patient and mouse PDAC revealed higher expression of Sema3E in tumor cells compared to normal cells. Interestingly, Sema3E expression was concentrated in the nuclei of tumor cells while it has been reported in current literature to be a secreted protein found in normal tissues. Sema3E-transduced PDAC cell lines Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher Sema3E expression in cell lysates and culture supernatant compared to control cells. Mia PaCa-2-Sema3E had a 60.4% higher rate of colony formation than the control (p = 0.005), while both Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher proliferation rates (58.8%; p Conclusions: Our findings indicate that Sema3E is overexpressed in PDAC and it may play some pathogenic roles in PDAC progression by enhancing tumor cell proliferation. These findings indicate that Sema3E could be an attractive novel therapeutic target in PDAC treatment strategies. Citation Format: Lin-Kin Yong, Dali Li, Ethan Poteet, Zhengdong Liang, William Fisher, Changyi Chen, Qizhi Cathy Yao. Novel roles of an axon-guidance molecule, semaphorin 3E, in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2015-3574