Search

// Tatsuo Matsuda 1 , Taigo Kato 1 , Kazuma Kiyotani 1 , Yunus Emre Tarhan 1 , Vassiliki Saloura 1 , Suyoun Chung 2 , Koji Ueda 3 , Yusuke Nakamura 1,4 and Jae-Hyun Park 1 1 Department of Medicine, The University of Chicago, Chicago, IL, USA 2 OncoTherapy Science Inc., Kawasaki, Japan 3 Project for Realization of Personalized Cancer Medicine, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan 4 Department of Surgery, The University of Chicago, Chicago, IL, USA Correspondence to: Yusuke Nakamura, email: // Keywords : maternal embryonic leucine zipper kinase, molecular target, p21, p53, FoxO family Received : April 12, 2017 Accepted : June 01, 2017 Published : June 15, 2017 Abstract MELK play critical roles in human carcinogenesis through activation of cell proliferation, inhibition of apoptosis and maintenance of stemness. Therefore, MELK is a promising therapeutic target for a wide range of cancers. Although p21 is a well-known p53-downstream gene, we found that treatment with a potent MELK inhibitor, OTS167, could induce p21 protein expression in cancer cell lines harboring loss-of-function TP53 mutations. We also confirmed that MELK knockdown by siRNA induced the p21 expression in p53-deficient cancer cell lines and caused the cell cycle arrest at G1 phase. Further analysis indicated that FOXO1 and FOXO3, two known transcriptional regulators of p21, were phosphorylated by MELK and thus be involved in the induction of p21 after MELK inhibition. Collectively, our herein findings suggest that MELK inhibition may be effective for human cancers even if TP53 is mutated.

The immune microenvironment of tumor plays a critical role in therapeutic responses to chemotherapy. Cancer tissues are composed of a complex network between anti-tumor and pro-tumor immune cells and molecules; therefore a comprehensive analysis of the tumor immune condition is imperative for better understanding of the roles of the immune microenvironment in anticancer treatment response. In this study, we performed T cell receptor (TCR) repertoire analysis of tumor infiltrating T cells (TILs) in cancer tissues of pre- and post-neoadjuvant chemotherapy (NAC) from 19 breast cancer patients; five cases showed CR (complete response), ten showed PR (partial response), and four showed SD/PD (stable disease/progressive disease) to the treatment. From the TCR sequencing results, we calculated the diversity index of the TCRβ chain and found that clonal expansion of TILs could be detected in patients who showed CR or PR to NAC. Noteworthy, the diversity of TCR was further reduced in the post-NAC tumors of CR patients. Our quantitative RT-PCR also showed that expression ratio of CD8/Foxp3 was significantly elevated in the post-NAC tumors of CR cases (p=0.0032), indicating that antitumor T cells were activated and enriched in these tumors. Collectively, our findings suggest that the clonal expansion of antitumor T cells may be a critical factor associated with response to chemotherapy and that their TCR sequences might be applicable for the development of TCR-engineered T cells treatment for individual breast cancer patients when their tumors relapse.

Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular roles of their aberrant glycosylations on cancer have not been fully understood. We previously reported critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6 or GalNAc-T6) that was upregulated in a great majority of breast cancer tissues. Here we further report O-glycosylation of estrogen receptor alpha (ER-α) by GALNT6 and the significant role of its nuclear localization in breast cancer cells. Knockdown of GALNT6 expression in two breast cancer cell lines, T47D and MCF7, in which both ER-α and GALNT6 were highly expressed, by small interfering RNA could significantly attenuate expression of ER-α. Immunocytochemical analysis clearly demonstrated the drastic decrease of ER-α protein in the nucleus of these cancer cells. Accordingly, the downstream genes of the ER-α pathway such as MYC, CCND1, and CTSD were significantly downregulated. We confirmed GALNT6-dependent ER-α O-glycosylation and identified O-glycosylation of S573 in an F domain of ER-α by GALNT6 through LC-MS/MS analysis. We also obtained evidences showing that the glycosylation of ER-α at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-α in breast cancer cells. Furthermore, we designed cell membrane–permeable peptides including the O-glycosylation site and found a significant decrease of the cell viability of breast cancer cells by treatment of these peptides in a GALNT6 expression–dependent manner. Our study suggests that targeting the GALNT6 enzymatic activity as well as the GALNT6/ER-α interaction could be a promising therapeutic approach to ER-α–positive breast cancer patients.

Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular mechanisms of such aberrant modifications and their effects on cancer development have not been fully understood. We previously reported the critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), which is upregulated in a great majority of breast cancers and is responsible for initiating mucin-type O-glycosylation. Suppression of GALNT6 expression by small interfering RNA to GALNT6 significantly enhanced cell-cell adhesion, induced mesenchymal-epithelial transition, and suppressed the growth of breast cancer cells. Here we further analyzed molecular functions of GALNT6 and found that GALNT6 could glycosylate an estrogen receptor alpha (ER-α) protein, a molecule playing a central role in proliferation of hormone-dependent breast cancer cells. We have used two breast cancer cell lines, T47D and MCF7, in which both the estrogen receptor and GALNT6 were highly expressed. Knockdown of GALNT6 expression by siRNA could significantly attenuate expression of ER-α at transcriptional and protein levels in these breast cancer cell lines in a condition with or without estradiol (P Citation Format: Boya Deng, Yunus Emre Tarhan, Koji Ueda, Yo Matsuo, Jae-Hyun Park, Yusuke Nakamura. Glycosylation of estrogen receptor alpha by N-acetylgalactosaminyltransferase 6 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 977.

Pancreatic Cancer has one of the lowest rates of survival among other cancers. Although molecular targeted therapy has been widely applied for many types of cancer, no effective treatment has been developed for the patients of pancreatic cancer. We previously suggested that GALNT6 (polypeptide N-acetylgalactosaminyltransferase 6) is a promising molecular target in breast cancer by O-type glycosylation of Mucin1. Interestingly, GALNT6 is also up-regulated in pancreatic cancer where Mucin proteins are intensively O-glycosylated, indicating a critical role of GALNT6 in pancreatic cancer. In this study we found that knockdown of GALNT6 using siRNAs decreased Muc4 protein as well as transcription level in pancreatic cancer cells. The knockdown of GALNT6 also decreased HER2 and ERK, interacting proteins of Mucin 4, accompanied by drastic reduction of pancreatic cancer cell viability. Further analysis revealed that knockdown of GALNT6 led to cell morphologic changes like the mesenchymal to epithelial transition, which was in concordant with the cadherin switching mechanism—increase of E-cadherin meanwhile decrease of P-cadherin level. Taking into considerations of important roles of Mucin 4 in growth, invasion and resistance to gemcitabine, our findings imply that targeting GALNT6 is a very promising therapeutic strategy for the treatment of pancreatic cancer patients who still have very limited treatment modalities Citation Format: Yunus Emre Tarhan, Kato Taigo, Miran Jang, Yusuke Nakamura, Jae-Hyun Park. Morphological changes, cadherin switching, and growth suppression in pancreatic cancer by GALNT6 knockdown. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B27.