Hyeong-Reh Choi Kim

Nuclear Export of Phosphorylated Galectin-3 Regulates Its Antiapoptotic Activity in Response to Chemotherapeutic Drugs

ABSTRACT Galectin-3 (Gal-3), a member of the β-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser6 by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser6 regulates Gal-3 function. We have generated serine-to-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser6 phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3.

TIMP-1 regulation of cell cycle in human breast epithelial cells via stabilization of p27KIP1 protein

Increasing evidence suggests that tissue inhibitor of metalloproteinases-1 (TIMP-1) can directly regulate cell growth and apoptosis independent of its matrix metalloproteinases (MMPs)-inhibitory activity. While TIMP-1s antiapoptotic activity has been well demonstrated, conflicting data has been reported regarding TIMP-1s role in growth regulation. Here we show that TIMP-1 reduces the growth rate of human breast epithelial (MCF10A) cells by inducing cell cycle arrest at G1. TIMP-1-mediated cell cycle arrest is associated with its downregulation of cyclin D1 and upregulation of p27KIP1, resulting in inhibition of cyclin-dependent kinase activity necessary for phosphorylation of the tumor suppressor retinoblastoma protein. We further show that TIMP-1 modulation of cyclin D1 and p27KIP1 is achieved through TIMP-1-mediated differential regulation of protein stability independent of growth factor signaling. We also show that TIMP-1-mediated differential regulation of cyclin D1 and p27KIP1 is independent of cell adhesion signaling. Whereas approximately 50% of MCF10A cells with reduced TIMP-1 expression underwent cell death following loss of cell adhesion (anoikis), TIMP-1 overexpressing cells remained viable with prominent cell cycle arrest without detectable cell death. Taken together, we propose that TIMP-1-mediated cell survival independent of cell adhesion is accompanied with cell cycle arrest in human breast epithelial cells, although cell cycle regulation may not be a prerequisite for TIMP-1 regulation of apoptosis in general.

A novel function for platelet-derived growth factor D: induction of osteoclastic differentiation for intraosseous tumor growth

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor β (β-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of β-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for β-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate β-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage

Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.

Matriptase activation and shedding through PDGF-D-mediated extracellular acidosis

Activation of β-platelet-derived growth factor receptor (β-PDGFR) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification, which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor hypoxia-inducible factor-1α (HIF-1α). Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D-expressing cells. Treatment of vector control or PDGF-B-expressing cells with the HIF-1α activator CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis, and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D-transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.

HPV-associated differential regulation of tumor metabolism in oropharyngeal head and neck cancer

HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. The goal of the present study is to investigate the molecular mechanisms underlying the differential radiation sensitivity between HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC). Here, we show that HPV-negative HNSCC cells exhibit increased glucose metabolism as evidenced by increased production of lactate, while HPV-positive HNSCC cells effectively utilize mitochondrial respiration as evidenced by increased oxygen consumption. HPV-negative cells express HIF1α and its downstream mediators of glucose metabolism such as hexokinase II (HKII) and carbonic anhydrase IX (CAIX) at higher levels, while the expression level of cytochrome c oxidase (COX) was noticeably higher in HPV-positive HNSCC. In addition, the expression levels of pyruvate dehydrogenase kinases (PDKs), which inhibit pyruvate dehydrogenase activity, thereby preventing entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle, were much higher in HPV-negative HNSCC compared to those in HPV-positive cells. Importantly, a PDK inhibitor, dichloroacetate, effectively sensitized HPV-negative cells to irradiation. Lastly, we found positive interactions between tonsil location and HPV positivity for COX intensity and COX/HKII index ratio as determined by immunohistochemical analysis. Overall survival of patients with HNSCC at the tonsil was significantly improved with an increased COX expression. Taken together, the present study provides molecular insights into the mechanistic basis for the differential responses to radiotherapy between HPV-driven vs. spontaneous or chemically induced oropharyngeal cancer.HPV-positive oropharyngeal cancer patients experience significantly lower locoregional recurrence and higher overall survival in comparison with HPV-negative patients, especially among those who received radiation therapy. The goal of the present study is to investigate the molecular mechanisms underlying the differential radiation sensitivity between HPV-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC). Here, we show that HPV-negative HNSCC cells exhibit increased glucose metabolism as evidenced by increased production of lactate, while HPV-positive HNSCC cells effectively utilize mitochondrial respiration as evidenced by increased oxygen consumption. HPV-negative cells express HIF1α and its downstream mediators of glucose metabolism such as hexokinase II (HKII) and carbonic anhydrase IX (CAIX) at higher levels, while the expression level of cytochrome c oxidase (COX) was noticeably higher in HPV-positive HNSCC. In addition, the expression levels of pyruvate dehydrogenase kinases (PDKs), which inhibit pyruvate dehydrogenase activity, thereby preventing entry of pyruvate into the mitochondrial tricarboxylic acid (TCA) cycle, were much higher in HPV-negative HNSCC compared to those in HPV-positive cells. Importantly, a PDK inhibitor, dichloroacetate, effectively sensitized HPV-negative cells to irradiation. Lastly, we found positive interactions between tonsil location and HPV positivity for COX intensity and COX/HKII index ratio as determined by immunohistochemical analysis. Overall survival of patients with HNSCC at the tonsil was significantly improved with an increased COX expression. Taken together, the present study provides molecular insights into the mechanistic basis for the differential responses to radiotherapy between HPV-driven vs. spontaneous or chemically induced oropharyngeal cancer.

Abstract A33: The regulation of extracellular acidosis and cell survival by the TIMP-1/CD63 axis in breast carcinoma

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Breast Cancer (BrCa) resistance to conventional therapies often leads to more aggressive and metastatic disease highlighting the importance of understanding the resistance pathway. The Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is among the most reliable prognostic markers for therapy resistance as well as metastasis of breast cancer via promotion of breast cancer cell survival through its unique interaction with the tetraspanin CD63. Recent work has shown that levels of TIMP-1 and the carbonic anhydrase family member CAIX in circulating tumor cells (CTCs) predict BrCa progression-free and overall survival. Since CAIX is known to play a critical role in the regulation of extracellular acidosis that contributes to therapy resistance, we investigated whether TIMP-1/CD63 signaling regulates extracellular acidosis involving CAIX. We utilized the murine 4T1 and human MCF10A progression models of breast cancer and demonstrated increased CAIX and TIMP-1 expression in the more aggressive 4T1 and MCF10CA1h cells. When we monitored pH change using a digital pH reader and the pH sensitive DF fluorescein dye, the more aggressive human MCF10Ca1h cells exhibited marked extracellular acidosis compared to the minimally malignant MCF10A and MCF10AneoT cells. Importantly, downregulation of TIMP-1 or CD63 in MCF10CA1h cells specifically reduced CAIX expression and its associated extracellular acidosis. To determine the functional significance of CAIX in TIMP-1/CD63-mediated extracellular acidosis, we attenuated CAIX expression and demonstrated a significant reduction of the acidosis phenotype observed in MCF10CA1h cells. Importantly, CAIX knockdown drastically reduced BrCa cell survival in a clonogenic cell survival assay. Taken together, we have identified a unique TIMP-1/CD63-mediated signaling pathway, resulting in extracellular acidosis and cell survival of BrCa cells involving CAIX. This study further provides salient information as to TIMP-1-specific function in breast cancer. Citation Format: Abdo J. Najy, Young Suk Jung, Hyeong-Reh Choi Kim. The regulation of extracellular acidosis and cell survival by the TIMP-1/CD63 axis in breast carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A33.

Abstract 4420: Matriptase-mediated proteolytic regulation of biological activity and spatial distribution of PDGF-D

The oncogenic roles of platelet-derived growth factor D (PDGF-D) and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. The latent full-length PDGF-D consists of N-terminal CUB domain, C-terminal growth factor domain, and hinge region in between. Matriptase processes the full-length PDGF-D dimer (FL-D) into PDGF-D growth factor domain dimer (GFD-D) in a step-wise manner, involving generation of hemidimer (HD), which is an intermediate product containing one full-length PDGF-D subunit and one growth factor domain subunit. In this study, we showed that HD acts as a dominant-negative ligand of PDGF/PDGFR signaling in fibroblasts whereas it has its own unique function in pre-osteoclasts. The regulation of PDGF-D9s activity by matriptase is biphasic as demonstrated by the event that active PDGF-D GFD-D can be further cleaved into a smaller and yet inactive form if the enzymatic action of matriptase persists. By mutagenesis analyses, we identified amino acid residues R 340 R 341 GR 343 A at the C-terminus of PDGF-D growth factor domain to be the matriptase cleavage site through which PDGF-D is inactivated. Based on the sequence analysis in comparison with the published crystal structure of PDGF-B, we suggest that R 340 R 341 GR 343 A is within the loop III of PDGF-D growth factor domain, a critical domain for its binding to the PDGF receptor. When the positively charged arginine residues of R 340 R 341 GR 343 A were mutated to alanine residues, PDGF-D GFD-D lost its biological activity in activating β-PDGFR. Further, we found that proteolytic processing of latent PDGF-D to remove CUB domain and hinge region is tightly linked with the storage of PDGF-D GFD-D within extracellular matrix (ECM) in vitro. These results indicate the increased affinity to the ECM from FL-D, HD, to GFD-D and also suggest that CUB domain and hinge region of PDGF-D prevent the sequestration of PDGF-D in ECM. Interestingly, the matriptase cleavage site R 340 R 341 GR 343 A within the growth factor domain is also critical for PDGF-D association with ECM. Taken together, the present study reports structural elements crucial to the biologically function of PDGF-D and its association with ECM, suggesting that proteolytic processing is a crucial mechanism for PDGF-D to establish its tissue gradient. Citation Format: Wei Huang, Hyeong-Reh C. Kim. Matriptase-mediated proteolytic regulation of biological activity and spatial distribution of PDGF-D. [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 4420. doi:10.1158/1538-7445.AM2014-4420

Abstract 5263: Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In an attempt to identify biomarkers and/or therapeutic targets for malignant breast cancer, the present study showed a comparative proteome profiling of invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein DS-20, a crucial regulator of H-Ras activation, as a potential marker for invasive breast cancer. DS-20 (amino acid residues 1-38) interacted with H-Ras (residues 166-189) in lipid rafts and their interaction was important for H-Ras activation. DS-20 knockdown inhibited H-Ras activation and invasion in Hs578T triple-negative breast cancer cells (TNBC) and T24 bladder carcinoma cells in which H-Ras is endogenously activated, suggesting a crucial role of DS-20 in the invasive program which relies on the activation of H-Ras. We further showed that DS-20 was required for epidermal growth factor-induced H-Ras activation, but not that of N-Ras, in MDA-MB-231 TNBC cells. Intravasation of MDA-MB-231 cells treated with shRNA DS-20 in the chick chorioallantoic membrane model was markedly reduced, indicating that DS-20 is required for an invasive capacity in vivo. In a xenograft mice tumor model, DS-20 was essential for in vivo tumor aggressiveness of Hs578T cells, suggesting DS-20 as a potential target for the treatment of breast cancer. Using human breast cancer samples, we provide clinical evidence for the tumorigenic potential of DS-20 and its association with H-Ras. Taken together, our findings provide a new insight into the molecular basis of Ras isoform-specific interplay with plasma membrane leading to cell invasion. Citation Format: Hae-Young Yong, Eun-Sook Kim, Minsoo Koh, Hwajin Son, You Rim Jeon, Jin-Sun Hwang, Myeong-Ok Kim, Yujin Cha, Wahn Soo Choi, Dong-Young Noh, Kyung-Min Lee, Ki-Bum Kim, Jae-Seon Lee, Hyung Joon Kim, Hong-Hee Kim, Eun Joo Kim, So Yeon Park, Hyeong-Reh Choi Kim, Aree Moon. Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness. [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 5263. doi:10.1158/1538-7445.AM2014-5263

Abstract A25: Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression

Purpose: To unveil multi-faceted roles of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in cancer progression, particularly, those apart from Matrix Metalloproteinase (MMP) inhibition. Introduction: Clinical studies show increased TIMP-1 to associate with poor prognoses in many types of cancer, contrary to its well-known anti-invasive activity via MMP inhibition. Consistent with clinical data, we have made novel findings that TIMP-1 induces epithelial cell survival signaling and epithelial-mesenchymal transition (EMT) phenotype independent of MMP-inhibitory activity. Importantly, TIMP-1 signaling is dependent on interaction with the tetraspanin CD63, on the cell surface. Interestingly, CD63 was previously shown to bind and induce endocytosis of Membrane-type 1 MMP (MT1-MMP) to the lysosome. We have recently shown that over-expression of TIMP-1 can lead to increased MT1-MMP expression/activity which contribute to the resultant EMT phenotype. However, it has not yet been clarified if TIMP-1 and MT1-MMP have cooperative or competitive interactions with CD63. Methods/Results: To map the interacting domains of TIMP-1, or MT1-MMP with CD63, we have utilized yeast-2-hybrid (Y2H) and protein complementation assay (PCA). For Y2H, the proteins were fused to complementary fragments of the GAL-4 transcription factor. For PCA, partner proteins were fused to complementary fragments of a luciferase enzyme. Our results indicate that the Large Extracellular Loop (LEL) of CD63 may be responsible for TIMP-1 interaction; while the N-terminus or else transmembrane domains interact with MT1-MMP. For TIMP-1, the C-terminal end of the protein shows importance for CD63 interaction; which our preliminary results show can be specifically interrupted by use of a blocking antibody. Interestingly, despite differences in direct binding sites, TIMP-1 and MT1-MMP display competitive and not mutual interaction with CD63 according to our preliminary results. Conclusions: Taken together, we present a novel finding that TIMP-1/CD63 interaction and MT1-MMP/CD63 interaction are not compatible in their occurrence with each other. And that TIMP-1 over-expression in cancer therefore may disrupt the MT1-MMP/CD63 complex which offers MT1-MMP protection from lysosomal degradation. The C-terminus of TIMP-1 is critical to CD63 interaction and may provide a non-MMP-inhibitory site for therapeutically targeting TIMP-1 in cancer. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule via CD63 interaction and as an indirect regulator of MT1-MMP, in addition to its well-known function as an inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression. [Support from: Ruth L. Kirschtein National Research Service Award T-32 CA009531, Thomas C. Rumble University Graduate Fellowship, and NIH/NCI RO1 089113] Citation Format: Richard B. Warner, Young-Suk Jung, Hyeong-Reh C. Kim. Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A25.