Matthew J. Schultz

Regulation of the metastatic cell phenotype by sialylated glycans

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines

The ST6Gal-I sialyltransferase adds an α2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here, we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations, we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.

ST6Gal-I sialyltransferase confers cisplatin resistance in ovarian tumor cells

BackgroundPlatinum drugs, including cisplatin, are a frontline therapeutic in ovarian cancer treatment and acquired resistance to these agents is a major contributor to ovarian cancer morbidity and mortality. In this study a novel glycosylation-dependent mechanism for cisplatin resistance is described. Specifically, cisplatin-induced cell death is blocked by the activity of the ST6Gal-I sialyltransferase. ST6Gal-I modifies specific receptors by adding a negatively charged sialic acid sugar which influences diverse receptor functions. Overexpression of ST6Gal-I is a hallmark of ovarian and other cancers and its expression has been correlated to metastasis and poor prognosis.MethodsTumor cell viability and apoptotic induction were determined in cell lines with ST6Gal-I overexpression and knockdown. In addition, cell populations with acquired resistance to cisplatin were assayed for endogenous ST6Gal-I expression.ResultsWe show that forced expression of ST6Gal-I in OV4 ovarian cancer cells that lack endogenous ST6Gal-I causes reduced activation of caspase 3 and increased cell viability following cisplatin treatment. Conversely, forced ST6Gal-I knockdown in Pa-1 cells with high endogenous ST6Gal-I increases cisplatin-induced caspase activation and cell death. A2780 ovarian cancer cells selected for stable cisplatin resistance display upregulated endogenous ST6Gal-I when compared with parental, cisplatin-sensitive, A2780 cells. Similarly, extended low dose cisplatin treatment of a Pa-1 polyclonal ST6Gal-I shRNA knockdown population led to selection for subclones with elevated ST6Gal-I expression.ConclusionsReceptor sialylation by ST6Gal-I confers a survival advantage for tumor cells in the presence of cisplatin. These collective findings support a role for ST6Gal-I in chemoresistance and highlight ST6Gal-I as a potential therapeutic target for platinum resistant tumors.

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.

The Tumor-Associated Glycosyltransferase ST6Gal-I Regulates Stem Cell Transcription Factors and Confers a Cancer Stem Cell Phenotype.

The glycosyltransferase ST6Gal-I, which adds α2-6-linked sialic acids to substrate glycoproteins, has been implicated in carcinogenesis; however, the nature of its pathogenic role remains poorly understood. Here we show that ST6Gal-I is upregulated in ovarian and pancreatic carcinomas, enriched in metastatic tumors, and associated with reduced patient survival. Notably, ST6Gal-I upregulation in cancer cells conferred hallmark cancer stem-like cell (CSC) characteristics. Modulating ST6Gal-I expression in pancreatic and ovarian cancer cells directly altered CSC spheroid growth, and clonal variants with high ST6Gal-I activity preferentially survived in CSC culture. Primary ovarian cancer cells from patient ascites or solid tumors sorted for α2-6 sialylation grew as spheroids, while cells lacking α2-6 sialylation remained as single cells and lost viability. ST6Gal-I also promoted resistance to gemcitabine and enabled the formation of stably resistant colonies. Gemcitabine treatment of patient-derived xenograft tumors enriched for ST6Gal-I-expressing cells relative to pair-matched untreated tumors. ST6Gal-I also augmented tumor-initiating potential. In limiting dilution assays, subcutaneous tumor formation was inhibited by ST6Gal-I knockdown, whereas in a chemically induced tumor initiation model, mice with conditional ST6Gal-I overexpression exhibited enhanced tumorigenesis. Finally, we found that ST6Gal-I induced expression of the key tumor-promoting transcription factors, Sox9 and Slug. Collectively, this work highlighted a previously unrecognized role for a specific glycosyltransferase in driving a CSC state. Cancer Res; 76(13); 3978-88. ©2016 AACR.

Abstract 2114: Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells

ST6Gal-I is a sialyltransferase that adds α2-6 linked sialic acids to cell surface proteins as they pass through the trans-Golgi. Sialic acids, being negatively charged, are able to alter the function of selected cell surface receptors, which leads to dysregulation of various downstream cellular pathways. Overexpression of ST6Gal-I has been observed in various cancers including ovarian and pancreatic cancer. We have previously shown that knockdown of ST6Gal-I expression increases ovarian cancer cell susceptibility to the chemotherapeutic drug cisplatin. In the present study we further investigate whether resistance to additional drugs such as gemcitabine, the front line treatment for pancreatic cancer, is affected by ST6Gal-I activity. MiaPaCa-2 and BxPC3 pancreatic cancer cell lines, having high ST6Gal-I expression, were employed for our studies. To elucidate the mechanistic role of ST6Gal-I in gemcitabine resistance we created stable ST6Gal-I knockdown lines of MiaPaCa-2 and BxPC3 cells. Gemcitabine induced cell death was more pronounced in the knockdown cell lines, indicated by heightened activation of caspase-3. Gemcitabine is metabolized to a nucleoside analogue, gemcitabine triphosphate, which induces apoptosis by promoting DNA damage. Increased single stand DNA damage in the knockdown cells was confirmed using alkaline comet assay. Gemcitabine treatment led to greater activation of DNA damage markers and response elements (γH2AX, phospho CHK1, phospho CHK2), along with increased levels of cleaved caspase-3 in the knockdown cells as compared to empty vector control cells. We next developed a stable gemcitabine resistant MiaPaCa-2 cell line by growing parental cells in gemcitabine. We selected for the population of cells that survived and were able to replicate in gemcitabine containing media. ST6Gal-I levels were found to be increased in the stable gemcitabine resistant lines relative to parental cell lines, suggesting that cells with high ST6Gal-I expression selectively survive gemcitabine treatment. By measuring the levels of genes involved in activation over those responsible for inactivation of gemcitabine, we were able to obtain a gemcitabine sensitivity predictive ratio for MiaPaCa-2 and BxPC3 ST6Gal-I knockdown, empty vector, parental and stable gemcitabine resistant cell lines. This ratio has been described previously in literature as a metric for gauging gemcitabine resistance. According to the ratios obtained, ST6Gal-I knockdown enhanced drug sensitivity whereas high expression of ST6Gal-I offered protection against gemcitabine induced apoptosis. Collectively these data indicate that upregulation of ST6Gal-I imparts tumor cell survival through prevention of gemcitabine induced DNA damage. Citation Format: Asmi Chakraborty, Matthew Schultz, Hoa Q. Trummell, James A. Bonner, Susan Bellis. Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells. [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 2114.

Abstract 3327: The tumor associated sialyltransferase ST6Gal-I promotes a cancer stem cell phenotype and upregulates stem-related transcription factors

Altered glycosylation is a key hallmark of tumor cells; still, the role of individual glycosyltransferases remains unclear. ST6Gal-I is a tumor-associated sialyltransferase which catalyzes the addition of a sialic acid sugar to substrate glycoproteins. Addition of the negatively-charged sialic acid by ST6Gal-I has been shown to alter receptor conformation, clustering, and surface retention, leading to changes in downstream signaling. In this study we assayed ST6Gal-I by immunohistochemistry and report the great majority of patient ovarian and pancreatic tumors express this enzyme. In contrast, the normal epithelium expresses minimal ST6Gal-I. Enzyme expression in ovarian cancers is enriched during metastasis and correlates with worse progression-free and overall survival. Recent evidence points to ST6Gal-I activity in stem/progenitor cells. In light of this, we investigated whether ST6Gal-I functionally promotes a cancer stem cell (CSC) phenotype, i.e. resistance to chemotherapy, survival as tumorspheroids, and ability to initiate tumors. We previously reported that ST6Gal-I activity confers resistance to cisplatin; we now show its activity additionally confers resistance to gemcitabine in pancreatic tumor cells. ST6Gal-I expressing cells are enriched in patient derived xenografts (PDX) treated with gemcitabine suggesting that these cells preferentially survive chemotherapy in vivo. In addition to chemoresistance, ST6Gal-I promotes the growth of pancreatic and ovarian cell lines in tumorspheroid culture. Moreover, ST6Gal-I expressing primary tumor cells isolated from ovarian cancer ascites or PDX tumors survive in tumorspheroid culture, whereas ST6Gal-I negative cells do not. Conversely, forced expression of ST6Gal-I protects tumor cells exposed to the ascites fluid milieu in vitro, while non-ST6Gal-I expressing cells succumb to this inflammatory environment. In a limiting dilution tumor initiating assay, ST6Gal-I expressing cells have a higher tumor incidence and form larger tumors compared to cells with ST6Gal-I knockdown. We next created a conditional mouse model with forced ST6Gal-I expression in the intestinal tract and used AOM-DSS chemically-induced carcinogenesis model to evaluate tumor formation. Compared with wildtype mice, ST6Gal-I knock-in mice have a greater tumor burden, evidenced by increased tumor number and area. As a novel mechanistic link beteween ST6Gal-I and the CSC phenotype, direct modulation of ST6Gal-I levels in tumor cells regulates the expression of stem-related transcription factors, Sox9 and Slug, implicated in tumor progression. The finding that a distinct glycosyltransferase governs the expression of key transcription factors highlights the tumor glycome as a driving factor in CSC behavior. Citation Format: Matthew J. Schultz, Andrew T. Holdbrooks, Asmi Chakraborty, William E. Grizzle, Charles N. Landen, Donald J. Buchsbaum, Michael G. Conner, Rebecca C. Arend, Karina J. Yoon, Chris A. Klug, Daniel C. Bullard, Robert A. Kesterson, Patsy G. Oliver, Amber K. O’Connor, Bradley K. Yoder, Susan L. Bellis. The tumor associated sialyltransferase ST6Gal-I promotes a cancer stem cell phenotype and upregulates stem-related transcription factors. [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 3327.

Abstract 3566: Glycosylation of the TNFR1 death receptor controls cell fate

Activation of the TNFR1 death receptor by TNFα can induce cell survival or cell death signaling, however, the mechanisms regulating this TNFR1 signaling switch are poorly understood. TNFα-induced apoptosis is reported to require higher-order clustering and internalization of activated TNFR1, whereas surface retention of the activated receptor has been shown to promote survival signaling mediated by NFκB and MAPKs. Studies from our group have identified a novel glycosylation-dependent mechanism that controls this signaling switch. Specifically, we have found that TNFR1 localization and activity are regulated by the addition of a distinct sugar, an α2-6 linked sialic acid, by the enzyme ST6Gal-I, a Golgi sialyltransferase whose expression is upregulated in many cancer types. The sialylation effect on TNFR1 was examined in epithelial and monocytic cancer cell lines with forced overexpression or knockdown of ST6Gal-I, as well as in primary monocytes obtained from mice with ST6Gal-I knock-in or knockout. Data from these models indicate that α2-6 sialylation of TNFR1 blocks TNFα-induced apoptosis, and the suggested mechanism underlying this inhibition of apoptosis is the sialylation-driven inhibition of TNFR1 oligomerization and internalization, as observed through immunoblotting, immunocytochemistry and flow cytometry. Considering sialylated TNFR1 is retained on the plasma membrane following activation, we hypothesize that ST6Gal-I functions to not only block TNFR1-mediated cell death but also divert the cell9s activity to favor survival. Supporting this hypothesis, cells with elevated levels of ST6Gal-I exhibit heightened expression and activation of many pro-survival factors, such as NFκB, MAPKs and AKT, in addition to decreased activation of caspase-3. Since levels of ST6Gal-I are elevated in multiple tumor cell populations, our group proposes that ST6Gal-I protects tumor cells against TNFα-induced apoptosis within the inflammatory tumor microenvironment. As preliminary support for this concept, peritoneal ascites fluid was collected from ovarian cancer (OC) patients, and the acellular, cytokine-rich fraction was incubated with OC cell lines with or without forced ST6Gal-I expression. Expression of ST6Gal-I conferred strong protection against cell death induced by soluble ascites. Based on these collective findings, we postulate that sialylation of TNFR1 by ST6Gal-I diverts the cellular response to TNFα from apoptosis to survival, providing a mechanism by which tumor cells can evade immune cell killing. Citation Format: Andrew Holdbrooks, Matthew J. Schultz, Zhongyu Liu, Daniel Bullard, Susan L. Bellis. Glycosylation of the TNFR1 death receptor controls cell fate. [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 3566.

Abstract 3820: Expression of ST6Gal-1 in colorectal cancer and patient prognosis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: ST6Gal-1 sialyltransferase, a type II membrane protein that catalyzes the transfer of sialic acid to the N-glycans of certain receptors, is upregulated in several cancers, including colorectal cancers (CRCs), and its levels correlate with metastatic potential. Therefore, we investigated its prognostic value in CRCs. Methodology: Formalin-fixed, paraffin-embedded CRC tissues from African Americans (Blacks) (n=50) and non-Hispanic Caucasians (Whites) (n=51) patients were evaluated for immunohistochemical (IHC) expression of ST6Gal-1. IHC expression was assessed using an anti-human ST6Gal-1 polyclonal antibody (R&D Systems) with antigen retrieval. The antibody-antigen complex was detected by the Immpact NovaRed system (Vector Labs). A semiquantitative immunostaining scoring system (ISS) on a scale of 0-4 was developed to assess the ST6Gal-1 expression, and a cutoff value (1.2 ISS) was determined by the bootstrap method. This value was used to classify tumors as low- or high-ST6Gal-I expressors. Previously, these tissues were evaluated for p53 nuclear accumulation (p53nac), an indicator of abnormal p53, by IHC. ST6Gal-1 expression levels were correlated with demographic and clinicopathologic features and with patient survival. Results: Increased ST6Gal-1 expression was observed in all CRCs as compared to their corresponding normal tissues. The overall frequency of CRCs with increased ST6Gal-1 expression was 80%; it was more commonly found in Blacks (88%, 44 of 50) than in Whites (70%, 36 of 51) (X2 p=0.031). Also, increased expression was associated with p53nac, and ST6Gal-1 expression was higher in CRCs that exhibited p53nac (87%, 42 of 48) relative to CRCs without p53nac (70%, 35 of 50) (X2 p = 0.034). Moreover, patients with CRCs with high ST6Gal-1 had lower median survival (29.3 months) relative to those with low ST6Gal-1 expression (58.5 months). Conclusions: These preliminary findings suggest that increased expression of ST6Gal-1 expression, found predominantly in Blacks patients and in CRCs with abnormal p53, is associated with poor patient survival. Citation Format: Trafina Jadhav, Saksham Narang, Jeehyun Bae, Matthew Schultz, Isam-Eldin Eltoum, Susan Bellis, Sejong Bae, Upender Manne. Expression of ST6Gal-1 in colorectal cancer and patient prognosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2014-3820

Abstract A78: Control of tumor cell survival mechanisms by a novel glycan-modifying enzyme

Altered surface glycosylation was one of the earliest known hallmarks of a tumor cell, however mechanistic insight into the role of glycans in regulating tumor cell phenotype remains limited. Among the subset of glycosyltransferases dysregulated in tumor cells, ST6Gal-I stands out as being consistently upregulated in a variety of different cancers. ST6Gal-I is a Golgi sialyltransferase that adds an a2-6-linked sialic acid, a negatively-charged sugar, to a select group of membrane receptors. Our work has focused on defining ST6Gal-I substrates and elucidating the function of these aberrantly-sialylated receptors in promoting aggressive tumor cell behaviors. Our prior studies showed that ST6Gal-I-directed receptor sialylation enhances ovarian tumor cell invasiveness, and also prevents tumor cell death induced by key apoptotic mediators including galectins, chemotherapy drugs (cisplatin), and certain death receptor activators. In new unpublished studies we find a novel role for ST6Gal-I in controlling downstream signaling from two death receptors, TNFR1 and Fas. Sialylation of Fas and TNFR1 by ST6Gal-I prevents ligand-induced internalization of these receptors, an event essential for apoptotic signaling. However, sialylation of TNFR1 and Fas does not simply shut off receptor activity, but instead diverts signaling toward survival pathways mediated by NFkB and MAPKs. This switch occurs as a consequence of sialylation-dependent retention of TNFR1 and Fas at the cell surface. Given that the ligands for TNFR1 and Fas (TNFa and FasL, respectively) are expressed predominantly by immune cells, we hypothesized that ST6Gal-I sialylation of these receptors may facilitate tumor cell survival within an inflammatory milieu, contributing to escape from immune surveillance. Modeling the tumor microenvironment, we exposed ovarian cancer cells either lacking or overexpressing ST6Gal-I to peritoneal ascites fluid harvested from ovarian cancer patients. Ascites is rich in cytokines including TNFa, sFasL and galectins. Forced expression of ST6Gal-I in ovarian cancer cells conferred protection against ascites-induced cell death, implicating a role for receptor sialylation in tumor cell survival during transcoelomic metastasis. In other unpublished studies, we conducted the first immunohistochemical analyses of ST6Gal-I expression in ovarian cancer, and determined that ST6Gal-I is upregulated in the majority of tumor specimens, including papillary serous and endometrioid carcinomas. Prior to our work, ST6Gal-I had only been measured at the mRNA level in ovarian cancer due to a lack of effective antibodies. To better understand ST6Gal-I9s influence on tumor cell phenotype, RNA sequencing/Ingenuity Pathway Analyses were performed in ovarian cancer cells with forced ST6Gal-I overexpression, and it was shown that ST6Gal-I upregulation induced activation of pathways associated with cancer stem cells, including the Wnt signaling axis. These results are consistent with our prior studies in colon tumor cells indicating that ST6Gal-I expression is particularly high in cancer stem cells. Taken together, our results suggest that upregulation of ST6Gal-I in ovarian carcinoma leads to abnormal hypersialylation of a select group of receptors, in turn conferring an invasive, apoptosis-resistant, and chemoresistant tumor cell phenotype. Citation Format: Matthew J. Schultz, Zhongyu Liu, Charles N. Landen, Susan L. Bellis. Control of tumor cell survival mechanisms by a novel glycan-modifying enzyme. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A78.