David C. Schmitt

miR-125b Functions as a Key Mediator for Snail-induced Stem Cell Propagation and Chemoresistance

Background: Snail plays an important role in chemoresistance, but the mechanism is still unclear. Results: Up-regulation of microRNA-125b through Wnt signaling by snail enriches cancer stem cells and increases chemoresistance. Conclusion: MicroRNA-125b is a key mediator for Snail-induced stem cell propagation and chemoresistance. Significance: We reveal a novel mechanism for Snail-induced stem cell maintenance and chemoresistance. Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/β-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

Multifaceted roles of HSF1 in cancer

Heat shock transcription factor 1 (HSF1) is the master regulator of the heat shock response. Accumulating evidence shows that HSF1 is overexpressed in a variety of human cancers, is associated with cancer aggressiveness, and could serve as an independent diagnostic or prognostic biomarker. In this review, we will provide an overview of the multifaceted roles of HSF1 in cancer, with a special focus on the four underlying molecular mechanisms involved. First, HSF1 regulates the expression of heat shock proteins (HSPs) including HSP90, HSP70, and HSP27. Second, HSF1 regulates cellular metabolism, including glycolysis and lipid metabolism. Third, HSF1 serves as a regulator of different signaling pathways, such as HuR-HIF-1, Slug, protein kinase C (PKC), nuclear factor-kappaB (NF-κB), PI3K-AKT-mTOR, and mitogen-activated protein kinase (MAPK) pathways. Finally, HSF1 regulates microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Overall, HSF1 plays many important roles in cancer via regulating cell proliferation, anti-apoptosis, epithelial-mesenchymal transition (EMT), migration, invasion, and metastasis and may be a potential therapeutic target for human cancers.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1a

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

ErbB2-intronic microRNA-4728: a novel tumor suppressor and antagonist of oncogenic MAPK signaling.

Although the role of the ErbB2/HER2 oncogene in cancers has been extensively studied, how ErbB2 is regulated remains poorly understood. A novel microRNA, mir-4728, was recently found within an intron of the ErbB2 gene. However, the function and clinical relevance of this intronic miRNA are completely unknown. Here, we demonstrate that mir-4728 is a negative regulator of MAPK signaling through directly targeting the ERK upstream kinase MST4 and exerts numerous tumor-suppressive properties in vitro and in animal models. Importantly, our patient sample study shows that mir-4728 was under-expressed in breast tumors compared with normal tissue, and loss of mir-4728 correlated with worse overall patient survival. These results strongly suggest that mir-4728 is a tumor-suppressive miRNA that controls MAPK signaling through targeting MST4, revealing mir-4728’s significance as a potential prognostic factor and target for therapeutic intervention in cancer. Moreover, this study represents a conceptual advance by providing strong evidence that a tumor-suppressive miRNA can antagonize the canonical signaling of its host oncogene.

Knockout of BRD7 results in impaired spermatogenesis and male infertility.

BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7−/−) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7−/− mice, whereas DNA damage repair genes were down−regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia.

Identification of candidate biomarkers for the early detection of nasopharyngeal carcinoma by quantitative proteomic analysis.

UNLABELLED Nasopharyngeal carcinoma (NPC) is a major head and neck cancer with high occurrence in Southeast Asia and southern China. To identify novel biomarkers for the early detection of NPC patients, 2D-DIGE combined with MALDI-TOF-MS analysis was performed to identify differentially expressed proteins in the carcinogenesis and progression of NPC using LCM-purified normal nasopharyngeal epithelial tissues and various stages of NPC biopsies. As a result, 26 differentially expressed proteins were identified, of which two proteins with sharp expressional changes in the carcinogenic process, ENO1 and CYPA, were validated by western blot analysis and identified as critical seed proteins in the functional network. Immunohistochemistry assay was further performed to detect the expression of the two proteins with a tissue microarray that included various stages of NPC tissues. The ability of these proteins to detect NPC early was evaluated via a receiver operating characteristic analysis. The results indicated that the combination of the two proteins could perfectly discriminate NNET and AH from stage I of NPC with high sensitivity and specificity, which is more effective than using either of the two proteins individually. In summary, the combination of ENO1 and CYPA can serve as potential molecular markers for the early detection of NPC. BIOLOGICAL SIGNIFICANCE NPC is a lethal malignancy that is most prevalent in Southeast Asia, and early detection and treatment are essential for the survival and good prognosis of NPC patients. In the present work, we identified 26 differentially expressed proteins in NNET, AH and different stages of NPC tissues by using 2D-DIGE combined with MALDI-TOF/TOF analysis. Of these proteins, the down-regulation of ENO1 and over-expression of CYPA were confirmed with a high-throughput tissue microarray that included various stages of NPC tissues via an IHC assay, and the results indicated that the combination of ENO1 and CYPA can serve as a potential molecular marker for the early detection of NPC.

SPLUNC1 is associated with nasopharyngeal carcinoma prognosis and plays an important role in all-trans-retinoic acid-induced growth inhibition and differentiation in nasopharyngeal cancer cells

Human SPLUNC1 can suppress nasopharyngeal carcinoma (NPC) tumor formation; however, the correlation between SPLUNC1expression and NPC patient prognosis has not been reported. In the present study, we used a large‐scale sample of 1015 tissue cores to detect SPLUNC1 expression and its association with patient prognosis. SPLUNC1 expression was reduced in NPC samples compared to nontumor nasopharyngeal epithelium tissues. Positive expression of SPLUNC1 in NPC predicted a better prognosis (disease‐free survival, P = 0.034; overall survival, P = 0.048). Coxs proportional hazards model revealed that SPLUNC1 could be a significant prognostic factor affecting disease‐free survival (P = 0.027). A cDNA micro‐array analyzed by significant analysis of micro‐array (SAM) and ingenuity pathway analysis (IPA) revealed that an indirect interaction existed between SPLUNC1 and retinoic acid (RA) in the cancer regulatory network. To further investigate the molecular mechanisms involved, we utilized several bioinformatics tools and identified 12 retinoid X receptors heterodimer binding sites in the promoter region of the SPLUNC1 gene. The transcriptional activity of the SPLUNC1 promoter was up‐regulated significantly by all‐trans‐retinoic acid (ATRA). SPLUNC1 and retinoic acid receptor expression were induced significantly by ATRA, and removal of ATRA led to a progressive loss of SPLUNC1 and retinoic acid receptor expression. ATRA inhibited proliferation and induced the differentiation of NPC cells. Interestingly, over‐expression of SPLUNC1 sensitized NPC cells to ATRA, whereas knockdown of SPLUNC1 in HNE1 cells increased cell viability. Under SPLUNC1 knockdown conditions, differentiation was reversed by ATRA treatment. We concluded that SPLUNC1 could potentially predict prognosis for NPC patients and play an important role in ATRA‐induced growth inhibition and differentiation in NPC cells.

Abstract 3370: Natural compound targeting metabolism: A new insight for the treatment of triple negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Triple-negative breast cancer (TNBC) is a highly diverse group associated with an aggressive phenotype, characterized by early relapse and chemotherapy as the only treatment option. Thus, there is an urgent need to identify alternative strategies for the treatment of TNBC. Use of aerobic glycolysis is important marker of cancer cells and thus monitoring and targeting metabolism in cancer cells is an expanding area of research. Lactate dehydrogenase (LDH) A and B are important enzymes in the metabolic pathway. Traditionally, LDH-A expression has been used to measure the glycolytic capacity of cancer cells however, more recently LDH-B has also been identified as an important biomarker of metabolism. LDH-A is over-expressed in breast cancer, while presence of LDH-B in tumors is contradictory. Higher expression of LDH-B has been reported in normal tissues, with lower expression also noted in breast and prostate tumors. Thus, clarification of the role of LDH-A and LDH-B in cancer metabolism is important in identifying potential targets for TNBC treatment. NF-κB plays an important role in modulating cancer metabolism through regulating the balance between glycolysis and mitochondrial respiration and is over-expressed in TNBCs. Thus, the focus of the present study is to determine the effect of an NF-KB inhibitor in altering cellular metabolism of aggressive breast cancer cells. Experimental Design: We measured the metabolic capacity of several breast cancer cell lines via measurement of mitochondrial membrane potential, ATP level and expression of LDH-A and LDH-B. We then evaluated the ability of the NF-kB inhibitor, panepoxydone (PP), to alter these markers of metabolism. Results: MCF-7 and MDA-MB-231 cells showed dose-dependent decreases in mitochondrial membrane potential, in the form of increased mitochondrial damage, along with decreased ATP levels, when treated with the NF-kB inhibitor, PP. Immunoblotting of LDH-A and LDH-B expression in MCF-7 and MDA-MB-231 cells showed high expression of LDH-A in both cell lines, while MDA-MB-231 cells had a higher level of LDH-A compared to LDH-B, MCF-7 cells had no LDH-B. When these cells were treated with PP, LDH-A levels was reduced in both cancer cell lines (3.5 -4 fold). Interestingly, elevated level of LDH-B was noticed in MDA-MB-231 cells (2 fold). We used Human mammary epithelial cells (HMEC) to check the basal level of LDH-A and LDH-B and almost similar level was observed. Furthermore, this increase in LDH-B was associated with decreased aggressiveness, as noted by decreased invasion and migration. Conclusion: All together this data highlights the role of targeting NF-kB in altering glycolysis through modulation of the membrane potential, ATP level and LDH-A and LDH-B levels. Additionally, it appears that simultaneous measurement, of both LDH-A and LDH-B, may be a more accurate predictor of the metabolic capacity of cancer cells and thus a reflection of their aggressiveness. Citation Format: Ritu Arora, David Schmitt, Steve McClellan, Ming Tan, Windy Dean-Colomb. Natural compound targeting metabolism: A new insight for the treatment of triple negative 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 3370. doi:10.1158/1538-7445.AM2014-3370

Preparation of polyclonal antibody highly specific for mouse BRD7 protein and its application

Heran Wang1, Ran Zhao1, Ming Zhou1*, Chi Guo1, Yukun Liu1, Shihe Jiang1, Xiayu Li2, Wei Xiong1, Jian Ma1, Shuping Peng1, Xiaoling Li1, David C. Schmitt3, Ming Tan3, and Guiyuan Li1* Cancer Research Institute, Central South University, Changsha 410078, China Department of Gastroenterology, The Third Xiang-Ya Hospital, Central South University, Changsha 410013, China Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA These authors contributed equally to this work. *Correspondence address. Tel: þ86-731-84805412; Fax: þ86-73184805383; E-mail: zhouming2001@163.com (M.Z.)/E-mail: ligy@xysm.net (G.L.)

Abstract LB-242: MicroRNA-125b, a key mediator for Snail-induced stem cell propagation and chemoresistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC MicroRNAs (miRNAs) are short ribonucleic acid molecules commonly deregulated in various tumors. Our previous studies have shown that miR-125b plays a major role in the chemoresistance of breast cancer. Snail, a transcription factor important in epithelial-mesenchymal transition, has been reported to be responsible for tumor metastasis and recurrence, however, the mechanism by which Snail exerts these effects is not understood. We report a novel mechanism in which upregulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells and confers chemoresistance. We found that Snail is upregulated in chemoresistant cancer cells. Overexpressing Snail increases miR-125b expression by activating the Wnt/beta-catenin/TCF4 axis, while silencing Snail decreases miR-125b expression. Snail activates Wnt signaling by interacting with beta-catenin/TCF4 to activate the expression of target genes. We discovered 5 TCF4 binding sites in the miR-125b promoter. The full length miR-125b promoter showed higher activity with Snail overexpression, and deletion of TCF4 binding sites decreased the promoter activity of miR-125b in a dose-dependent manner. Knockdown of TCF4 decreased miR-125b expression, which confirms that TCF4 is required for Snail to fully activate the miR-125b promoter. Bak1 is a pro-apoptosis molecule and confirmed target of miR-125b. Deregulation of Bak1 is important in cancer development and drug resistance. Since overexpression of Snail confers chemotherapeutic resistance, and Snail was upregulated in Paclitaxel-resistant cells, we examined whether Snail induces drug resistance through Bak1. We found low Bak1 expression in Paclitaxel-resistant cells, which have high expression of Snail. To verify whether Snail regulates Bak1 expression, we expressed Snail in Snail-low expressing cells and saw repression of Bak1 expression. To confirm, we knocked-down Snail in Snail-high expressing cells, which resulted in increased Bak1 expression. Overexpression of Snail or miR-125b increased the resistance of cancer cells to chemotherapy. Inhibiting miR-125b expression or restoring Bak1 expression re-sensitized cells to chemotherapy. These results indicate that Snail increases chemoresistance by repressing Bak1 through miR-125b. Overexpression of miR-125b significantly increases the cancer stem cell population, while depletion of miR-125b or rescue of Bak1 expression increases the non-stem cell population. These findings support that miR-125b functions as a key mediator in Snail-induced cancer stem cells and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming chemotherapy resistance. Work supported by: Vincent F. Kilborn Jr. Cancer Research Foundation, NIH Grant R01CA149646, and the Norwegian Hospitalet Legater Project 334003. Citation Format: Zixing Liu, Hao Liu, Shruti Desai, David C. Schmitt, Ming Zhou, Hung T. Khong, Kristine S. Klos, Steven McClellan, Oystein Fodstad, Ming Tan. MicroRNA-125b, a key mediator for Snail-induced stem cell propagation and chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2013-LB-242