James P. De Andrade

Regulation of epithelial-mesenchymal transition through SUMOylation of transcription factors.

Carcinoma cells can transition from an epithelial-to-mesenchymal differentiation state through a process known as epithelial-mesenchymal transition (EMT). The process of EMT is characterized by alterations in the pattern of gene expression and is associated with a loss of cell polarity, an increase in invasiveness, and an increase in cells expressing cancer stem cell (CSC) markers. The reverse process of mesenchymal-to-epithelial transition (MET) can also occur, though the transitions characterizing EMT and MET can be incomplete. A growing number of transcription factors have been identified that influence the EMT/MET processes. Interestingly, SUMOylation regulates the functional activity of many of the transcription factors governing transitions between epithelial and mesenchymal states. In some cases, the transcription factor is a small ubiquitin-like modifier conjugated directly, thus altering its transcriptional activity or cell trafficking. In other cases, SUMOylation alters transcriptional mechanisms through secondary effects. This review explores the role of SUMOylation in controlling transcriptional mechanisms that regulate EMT/MET in cancer. Developing new drugs that specifically target SUMOylation offers a novel therapeutic approach to block tumor growth and metastasis.

Inhibiting the SUMO Pathway Represses the Cancer Stem Cell Population in Breast and Colorectal Carcinomas

Summary Many solid cancers have an expanded CD44+/hi/CD24−/low cancer stem cell (CSC) population, which are relatively chemoresistant and drive recurrence and metastasis. Achieving a more durable response requires the development of therapies that specifically target CSCs. Recent evidence indicated that inhibiting the SUMO pathway repressed tumor growth and invasiveness, although the mechanism has yet to be clarified. Here, we demonstrate that inhibition of the SUMO pathway repressed MMP14 and CD44 with a concomitant reduction in cell invasiveness and functional loss of CSCs in basal breast cancer. Similar effects were demonstrated with a panel of E1 and E3 SUMO inhibitors. Identical results were obtained in a colorectal cancer cell line and primary colon cancer cells. In both breast and colon cancer, SUMO-unconjugated TFAP2A mediated the effects of SUMO inhibition. These data support the development of SUMO inhibitors as an approach to specifically target the CSC population in breast and colorectal cancer.

EGFR Is Regulated by TFAP2C in Luminal Breast Cancer and Is a Target for Vandetanib

Expression of TFAP2C in luminal breast cancer is associated with reduced survival and hormone resistance, partially explained through regulation of RET. TFAP2C also regulates EGFR in HER2 breast cancer. We sought to elucidate the regulation and functional role of EGFR in luminal breast cancer. We used gene knockdown (KD) and treatment with a tyrosine kinase inhibitor (TKI) in cell lines and primary cancer isolates to determine the role of RET and EGFR in regulation of p-ERK and tumorigenesis. KD of TFAP2C decreased expression of EGFR in a panel of luminal breast cancers, and chromatin immunoprecipitation sequencing (ChIP-seq) confirmed that TFAP2C targets the EGFR gene. Stable KD of TFAP2C significantly decreased cell proliferation and tumor growth, mediated in part through EGFR. While KD of RET or EGFR reduced proliferation (31% and 34%, P < 0.01), combined KD reduced proliferation greater than either alone (52% reduction, P < 0.01). The effect of the TKI vandetanib on proliferation and tumor growth response of MCF-7 cells was dependent upon expression of TFAP2C, and dual KD of RET and EGFR eliminated the effects of vandetanib. The response of primary luminal breast cancers to TKIs assessed by ERK activation established a correlation with expression of RET and EGFR. We conclude that TFAP2C regulates EGFR in luminal breast cancer. Response to vandetanib was mediated through the TFAP2C target genes EGFR and RET. Vandetanib may provide a therapeutic effect in luminal breast cancer, and RET and EGFR can serve as molecular markers for response. Mol Cancer Ther; 15(3); 503–11. ©2016 AACR.

The Role of Staging Laparoscopy and Peritoneal Cytology in Gastric Cancer

Staging laparoscopy is an important yet underutilized tool in the treatment of patients with gastric adenocarcinoma. The principle goal of staging laparoscopy is to identify patients with operable primary lesions absent of macroscopic or microscopic metastatic disease. Despite appropriate preoperative radiographic staging, approximately a quarter of the patients with seemingly operable disease are in fact found to have occult gross metastatic lesions on staging laparoscopy. Moreover, patients with advanced lesions (serosal invasion, nodal disease) are particularly at a high risk of microscopic metastases into the surrounding peritoneal fluid. Sampling the peritoneal fluid for free tumor cells during laparoscopy identifies patients with microscopic metastatic disease, a diagnosis that portends a similarly poor prognosis to patients with gross metastatic disease. Identifying these patients prior to laparotomy not only saves the patient the morbidity of a needless major surgery but also prevents the delay of further chemotherapy.

Abstract P6-02-03: SUMO Inhibitors affect tumorigenesis of novel breast cancer xenograft model

A novel basal breast cancer cell line IOWA-1T was derived from chemotherapy resistant locally advanced breast cancer tumor. The cells rapidly form large, skin-eroding xenografts in nude mice. The SUMO inhibitor anacardic acid (AA) effectively cleared CD44+/hi/CD24-/low cancer stem cell (CSC) population in IOWA-1T and BT-20 basal cancer cell lines and delayed tumor outgrowth of basal cancer xenografts. The effect of SUMO inhibitors to clear the CSC population was dependent upon the SUMO unconjugated form of TFAP2A (Bogachek MV et al, Cancer Cell, 2014). Herein we show that tumors that eventually form from IOWA-1T xenografts in mice treated with AA are not capable of developing secondary xenografts, confirming eradication of the CSC population by SUMO inhibitors. As further mechanistic evidence for the SUMO pathway, transient knockdown of UBC9 and PIAS1 SUMOylation enzymes repressed CD44 expression and increased tumor free and overall survival in mice inoculated with IOWA-1T xenografts. Furthermore, CD44 downregulation was demonstrated in IOWA-1T cells after treatment in vitro with UBC9 inhibitor PYR-41 and PIAS1 inhibitor NSC-207895. Overall survival of mice with IOWA-1T xenografts was increased to 43±0.5 and 39±2 days with PYR-41 and NSC-207895 i.p. injections, respectively, compared to a vehicle treated control group 33±1 days (p Citation Format: Maria V Bogachek, Jung M Park, James P De Andrade, Mikhail V Kulak, Jeffrey R White, Tong Wu, Philip M Spanheimer, George W Woodfield, Thomas B Bair, Alicia K Olivier, Ronald J Weigel. SUMO Inhibitors affect tumorigenesis of novel breast cancer xenograft model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-03.

Targeting the sumoylation pathway in cancer stem cells

Cancer stem cells (CSCs) represent a subset of tumor cells with tumor-initiating potential. We recently demonstrated that inhibition of the sumoylation pathway cleared the CSC population and repressed the outgrowth of basal breast cancer xenografts. Targeting the sumoylation pathway offers a novel treatment strategy for basal breast cancer.

Abstract 3914: Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA TFAP2C and mouse homolog tcfap2c are transcription factors that play a critical role in mammary gland development and in maintaining the luminal breast cancer phenotype. Interestingly, several human HER2-positive breast cancer cell lines display genetic dependency on TFAP2C. However, the role of TFAP2C in carcinogenesis and progression in neu-activated breast cancer remains unknown. We examined the role of tcfap2c in mammary gland carcinogenesis by examining tumor formation in mice expressing an activated rat neu gene using MMTV-neu with and without mammary gland conditional knockout (KO) of tcfap2c using MMTV-Cre. The time until spontaneous palpable tumor formation was measured in mice harboring the neu oncogene with and without tcfap2c. Mice with homozygous conditional deletion of tcfap2c demonstrated a significant delay in tumor formation compared to littermate controls that retained tcfap2c expression (42 vs. 25 weeks, p<0.004). Immunohistochemistry of the tumors demonstrated a luminal phenotype; cytokeratin 8 (CK8), a luminal marker, was more strongly expressed compared to cytokeratin 5 (CK5), a basal marker, in tumors from both control (883 vs. 0.1 cells/HPF, p<0.0005) and KO animals (845 vs. 0, p<0.0005). Cleaved caspase-3 (CC3) showed that apoptosis was increased in tumors from KO compared to control animals (5 vs. 0.3 CC3-positive cells/HPF, p<0.0008). Ki-67 showed that proliferation was decreased in KO compared to control (348 vs. 142 cells/HPF, p<0.009). To validate the effects of tcfap2c, we established a cell line from a tumor obtained from an animal expressing activated neu with homozygous floxed tcfap2c lacking Cre expression. Paired cell lines with differential expression of tcfap2c were established by infecting the cells with Adenovirus-Cre (Ad-Cre) or Adenovirus-Empty (Ad-Empty). Proliferation was assessed by MTT and demonstrated that loss of tcfap2c expression resulted in a 30% decrease in proliferation rate (p<0.006). The difference in proliferation was confirmed in vivo through xenograft experiments of these paired cell lines inoculated into athymic (nude) mice. Mice xenografted with the paired cell lines demonstrated that KO of tcfap2c resulted in a significant delay in tumors reaching a 2 cm threshold requiring euthanasia (27.8 vs. 19.8 days post-inoculation, p<0.003). In order to identify a potential mechanism accounting for the differences in proliferation and delay in carcinogenesis, we identified tcfap2c target genes by comparing the binding sites of tcfap2c by chromatin-immunoprecipitation-sequencing (ChIP-seq) and differential expression by microarray/RT-PCR. We identified erbb3 and egfr as two tcfap2c target genes by ChIP-seq. KO of tcfap2c resulted in a 3-fold and 12-fold reduction in erbb3 and egfr expression, respectively (p<0.05). Taken together, these findings indicate that tcfap2c plays critical roles in oncogenesis and progression of HER2-positive mammary cancer. Citation Format: Jung M. Park, Tong Wu, George Woodfield, Anthony Cyr, James De Andrade, Weizhou Zhang, Frederick Domann, Ronald J. Weigel. Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer. [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 3914. doi:10.1158/1538-7445.AM2014-3914