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Background Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. Methods We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. Results SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. Conclusions Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone HJURP was aberrantly overexpressed in t(4;14)-positive multiple myeloma due to transcriptional activation by a distal SE induced by the histone lysine methyltransferase NSD2. Silencing of HJURP with short hairpin RNA or CRISPR interference of SE function impaired cell viability and led to apoptosis. Conversely, HJURP overexpression promoted cell proliferation and abrogated apoptosis. Mechanistically, the NSD2/BRD4 complex positively coregulated HJURP transcription by binding the promoter and active elements of its SE. In summary, this study introduces SE profiling as an efficient approach to identify new targets and understand molecular pathogenesis in specific subtypes of cancer. Moreover, HJURP could be a valuable therapeutic target in patients with t(4;14)-positive myeloma. SIGNIFICANCE: A super-enhancer screen in t(4;14) multiple myeloma serves to identify genes that promote growth and survival of myeloma cells, which may be evaluated in future studies as therapeutic targets. Published version The work was supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centres of Excellence initiative (to W.J. Chng), the NMRC Clinician Scientist Investigator award (to W.J. Chng), the RNA Biology Center at CSI Singapore, NUS, from funding by the Singapore Ministry of Education’s Tier 3 grants, grant number MOE2014-T3–1-006 (to W.J. Chng), the National Natural Science Foundation of China (grant no. 82000212 to Y. Jia), Natural Science Foundation of Zhejiang Province (grant no. LQ21H160022 to Y. Jia), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (grant no. 2021RC003 to Y. Jia). Y. Jia also thanks the China Scholarship Council (grant no. 201706320167) for financial support to visit National University of Singapore.

Multiple myeloma (MM) is an aggressive plasma cell neoplasm characterized by genomic heterogeneity. Superenhancers (SEs) are defined as large clusters of enhancers in close genomic proximity, which regulate genes for maintaining cellular identity and promote oncogenic transcription to which cancer cells highly addicted. Here, we analyzed cis-regulatory elements in MM samples with H3K27ac ChIP-seq, to identify novel SE-associated genes involved in the myeloma pathogenesis. SEs and their associated genes in cancerous tissue were compared with the control samples, and we found SE analysis alone uncovered cell-lineage-specific transcription factors and well-known oncogenes ST3GAL6 and ADM. Using a transcriptional CDK7 inhibitor, THZ1, coupled with H3K27ac ChlP-seq, we identified MAGI2 as a novel SE-associated gene of myeloma cells. Elevated MAGI2 was related to myelomagenesis with gradual increased expression from MGUS, SMM to newly diagnosed and relapsed MM. High prevalence of MAGI2 was also associated with poor survival of MM patients. Importantly, inhibition of the SE activity associated with MAGI2 decreased MAGI2 expression, inhibited cell growth and induced cell apoptosis. Mechanistically, we revealed that the oncogenic transcription factor, MAF, directly bound to the SE region and activated gene transcription. In summary, the discoveries of these acquired SEs-associated genes and the novel mechanism by which they are regulated provide new insights into MM biology and MAGI2-MAF-SE regulatory circuit offer potential novel targets for disease treatment.

Cholangiocarcinoma (CCA) is a group of malignant heterogeneous cancer arising from the biliary tree. CCA has become a global health problem with rising incidence and mortality that threatens the health of human beings. The immune microenvironment of CCA is characterized by abundant cancer-associated fibroblast and suppressive immune components. The increasing body of knowledge and recent developments in transcriptomic studies have given insight into the immune landscape of CCA, paving the way for better application of immunotherapy. Immunotherapy mainly applies in a limited subset of CCA with deficient mismatch and high microsatellite instability. With limited response rates and treatment efficacy, researchers are looking into novel strategies on combination strategies and alternatives, such as immune vaccines and adoptive cell therapy. Biomarker identification is also critical for patient selection. We present an up-to-date summary of the current research on immunotherapy for CCA patients, covering pre-clinical and clinical exploration beyond immune checkpoint inhibitors, immune vaccines, and adoptive cell therapy. In addition, we review the promising biomarkers for CCA immunotherapy and discuss recent development.

Chemoresistance is a daunting challenge to the prognosis of patients with breast cancer. Signal transducer and activator of transcription (STAT) 5a plays vital roles in the development of various cancers, but its function in breast cancer is controversial, and its role in chemoresistance in breast cancer remains unexplored. Here we identified STAT5a as a chemoresistance inducer that regulates the expression of ABCB1 in breast cancer and can be targeted by pimozide, an FDA-approved psychotropic drug. First, we found that STAT5a and ABCB1 were expressed at higher levels in doxorubicin-resistant cell lines and chemoresistant patients, and their expression was positively correlated. Then, we confirmed the essential roles of STAT5a and ABCB1 in doxorubicin resistance in breast cancer cells and the regulation of ABCB1 transcription by STAT5a. Subsequently, the efficacy of pimozide in inhibiting STAT5a and sensitizing doxorubicin-resistant breast cancer cells was tested. Finally, we verified the role of STAT5a in doxorubicin resistance in breast cancer and the efficacy of pimozide in reversing this resistance in vivo. Our study demonstrated the vital role of STAT5a in doxorubicin resistance in breast cancer. Targeting STAT5a might be a promising strategy for treating doxorubicin-resistant breast cancer. Moreover, repurposing pimozide for doxorubicin resensitization is attractive due to the safety profile of pimozide.

Background Growing evidence suggests that alterations in epigenetic landscape contribute to pathogenesis of multiple myeloma (MM). MM cells are highly dependent on unfolded protein response signaling pathways due to high level of endoplasmic reticulum stress. Phosphorylation of eIF2a can attenuate protein translation. The PPP1R15B(denoted as R15B hereafter) gene encodes a regulatory subunit of eIF2a-specific phosphatase complex. In this study,we identified super enhancer (SE)-driven oncogenes specific in MM with a particular focus on a candidate gene R15B, whose functional roles in MM remain largely elusive. Methods We performed H3K27Ac ChIP-seq on MM cell lines, primary MM patient samples, normal CD138+ plasma cells and memory B cells. ROSE analysis was used to annotate SEs and their associated genes. A combination of public data mining, RNAi, overexpression and CRISPR/Cas9 technologies were conducted to determine the oncogenic effects of R15B in MM. Transcriptome analysis of MM cell line H929 with R15B KD and scrambled control was performed. To further study the interactions between SE and its promoters, we are currently working on HiChIP. Results We have identified R15B as one of the SE-associated genes specific to MM patient samples and cell lines. SE activity was correlated with the expression level of R15B. Higher expression of R15B predicted poor overall survival of MM patients, suggesting its clinical relevance in MM pathogenesis. R15B KD or KO significantly reduced cell viability, clonogenicity and induced G2/M arrest. ChIP-qPCR assays showed that C/EBP-β is strongly enriched at R15B SE region. We also found that salubrinal, a selective inhibitor of eIF2a phosphorylation, inhibited MM cell proliferation in a dose-dependent manner. Conclusions Our integrative approaches identified R15B as a novel SE-driven oncogene. We propose that targeting R15B may serve as a new approach for effective anti-myeloma therapy, which warrants further clinical investigation.

Background Multiple myeloma (MM) is an incurable malignancy characterized by complex heterogeneous cytogenetic abnormalities. Defined as large clusters of cis-acting enhancers, super-enhancers (SEs) could promote oncogenic transcription to which cancer cells highly addicted. Structural maintenance of chromosome 4 (SMC4) is a core subunit of condensin complexes, which contributes to chromosome condensation and segregation. Here, we identified SMC4 as a novel SE-associated oncogene of myeloma cells, whose functional roles in MM remain largely elusive. Methods We performed H3K27ac ChIP-seq and RNA-seq on primary MM patient samples, MM cell lines, normal CD138+ plasma cell and lymphoma cell lines as the controls, then the ROSE analysis was used to annotate SEs and their associated genes. THZ1 as a transcriptional CDK7 inhibitor was used to further filter THZ1-responsive genes. Combined analysis of THZ1-sensitive and SE-associated gene uncovered a number of promising MM oncogenes as SMC4. The public data mining, RNA interference, CRISPR/Cas9 gene editing system, overexpression and a variety of cellular functional assays were performed to determine the oncogenic effects of SMC4 on MM malignant. Transcriptome analysis with SMC4 silencing and overexpression, and the underlying molecular mechanism of SMC4 oncogenic role in MM are ongoing. Results We have demonstrated SMC4 as one of the SE-associated genes specific to MM patient samples and cell lines. SMC4 was related to myelomagenesis with increased expression from MGUS, SMM to MM cases, and overexpression of SMC4 predicted poor overall survival of MM patients, suggesting its clinical relevance. SMC4 silencing in MM cells suppressed cell proliferation and lead to cell apoptosis. Repression of the SMC4-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function. Conclusion In summary, our integrative approached by combing H3H27ac ChIP-seq, RNA-seq and THZ1-sensitive transcripts identified the landscape of SEs and novel oncogenic transcripts as SMC4 in MM. Mapping these acquired SEs and their associated genes may provide novel insight into understanding MM biology, and SMC4 may serve as novel therapeutic targets in MM. Citation Format: Yunlu Jia, Jianbiao Zhou, Tze-King Tan, Tae-Hoon Chung, Yongxia Chen, Enfan Zhang, Zhen Cai, Takaomi Sanda, Wee-Joo Chng. Superenhancer-drvien SMC4 promote cell growth and proliferation in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2956.

Long non‑coding RNA (lncRNA) H19 and Lin28 protein have been shown to participate in various pathophysiological processes, including cellular proliferation, autophagy and epithelial‑mesenchymal transition (EMT). A number of studies have investigated lncRNAs, microRNAs and mRNAs, and their roles in the initiation and progression of cancer, in doing so identifying competitive endogenous RNA (ceRNA) networks, including the H19/let‑7/Lin28 network. However, whether the H19/let‑7/Lin28 ceRNA network is involved in autophagy and EMT in breast cancer (BC) remains unclear. The present study demonstrated that the H19/let‑7/Lin28 loop was required for the downregulation of autophagy in BC cells via western blot analysis, reverse transcription‑quantitative PCR and autophagy flux monitoring. Using wound healing, migration and invasion assays, and morphological assays, the H19/let‑7/Lin28 loop was revealed to promote EMT in BC cells. Moreover, the H19/let‑7/Lin28 network was found to contribute to autophagy by inhibiting EMT in BC cells. To the best of our knowledge, the present study is the first to suggest the important roles of the H19/let‑7/Lin28 ceRNA network in BC autophagy and EMT, thus providing insight for the use of these molecules as prognostic biomarkers and therapeutic targets in BC metastasis.

Matrix metalloproteinase-1 (MMP1) is a member of the matrix metalloproteinases family, and its aberrant expression is implicated in tumor invasion and metastasis. However, the relationship between MMP1 abnormal expression and clinical outcome in breast cancer patients remains to be elucidated. To address this issue, we conducted immunohistochemistry in breast cancer and adjacent normal tissues, and mined the transcriptional and survival data of MMP1 in breast cancer patients through Oncomine, Kaplan-Meier Plotter, bc-GenExMiner, COSMIC and cBioPortal databases. First, we found that both protein and mRNA levels of MMP1 expression were significantly higher in breast cancer tissues. Second, high MMP1 mRNA expression correlated with worse overall survival among grade II (HR = 1.75; p = 0.011), nodal-negative (HR = 2.00; p = 0.00028), ER-positive (HR = 1.61; p = 0.00027) and HER2-negative (HR = 3.17; p = 0.029) patients with breast cancer by using Kaplan-Meier plotter database. Third, the overexpression of MMP1 was associated with unfavorable survival results including overall survival (HR = 1.6; p = 1.6e-05), relapse free survival (HR = 1.78; p < 1e-16) and distant metastasis free survival (HR = 1.65; p = 5.3e-05) in patients with breast cancer. Taken together, the expression status of MMP1 is a significant prognostic indicator and a potential drug target for breast cancer.

// Xiao Luo 1, * , Yerfan Jiaerken 1, * , Xinfeng Yu 1 , Peiyu Huang 1 , Tiantian Qiu 1 , Yunlu Jia 3 , Kaicheng Li 1 , Xiaojun Xu 1 , Zhujing Shen 1 , Xiaojun Guan 1 , Jiong Zhou 2 and Minming Zhang 1 , for The Alzheimer’s Disease Neuroimaging Initiative (ADNI) 1 Department of Radiology, The Second Affiliated Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China 2 Department of Neurology, The Second Affiliated Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China 3 Department of Surgical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China * These authors have contributed equally to this work Correspondence to: Minming Zhang, email: zhangminming@zju.edu.cn Keywords: apolipoprotein E (APOE), cerebral small-vascular disease (CSVD), white matter hyperintensities (WMH), dilated perivascular space (dPVS), cognition Abbreviations: APOE: apolipoprotein E; CSVD: cerebral small-vascular disease; WMH: white matter hyperintensities; dPVS: dilated perivascular space; MBs: microbleeds Received: December 29, 2016 Accepted: April 07, 2017 Published: May 09, 2017 ABSTRACT Objective: It remains unclear if and how the interactions between APOE genotypes and cerebral small-vessel diseases (CSVD) lead to cognitive decline in the long term. Based on ADNI cohort, this longitudinal study aimed to clarify the potential relationship among APOE genotype, CSVD and cognition by integrating multi-level data. Method: There were 135 healthy elderly (including e2, e4 allele carriers and e3 homozygotes) who had completed two years’ follow-up. MRI markers of CSVD, including white matter hyperintensities (WMH), dilated perivascular space (dPVS), microbleeds and lacune, were assessed. Besides, neuropathological factors including Alzheimer’s disease-related pathology measured by CSF and PiB-PET were assessed. Repeated measurements ANOVAs were performed to test impact of different APOE genotypes on CSVD. Results: We found that APOE e4 carriers had significantly more frontal WMH burden and basal ganglia dPVS at baseline and faster progression of frontal WMH burden during follow-up. Furthermore, our results showed that APOE e4 carriers had significantly decreased Aβ 1-42 level, and its level was negatively related with baseline and progressive total WMH burden. Then, general linear modals indicated interaction between basal frontal WMH burden and e4 allele was related with declining trend of cognition. Conclusion: Our findings suggested APOE e4 allele was associated with increased Aβ deposition, which may lead to the formation and progression of WMH, especially in frontal lobe. Besides, interaction between the increased frontal WMH burden and e4 allele can exert long-term detrimental effects on individual’s trajectory of cognition.

// Hanchu Xiong 1,2,* , Wenhe Zhao 1,2,* , Ji Wang 1,2,* , Benjamin J. Seifer 3 , Chenyang Ye 4 , Yongxia Chen 1,2 , Yunlu Jia 1,2 , Cong Chen 1,2 , Jianguo Shen 1,2 , Linbo Wang 1,2 , Xinbing Sui 2,5 and Jichun Zhou 1,2 1 Department of Surgical Oncology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China 2 Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, Zhejiang, China 3 USF Health Morsani College of Medicine, Tampa, FL, USA 4 Cancer Institute (Key Laboratory of Cancer Prevention & Intervention, National Ministry of Education), Second Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China 5 Department of Medical Oncology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, Zhejiang, China * These authors have contributed equally to this work Correspondence to: Linbo Wang, email: // Xinbing Sui, email: // Jichun Zhou, email: // Keywords : Lin28; Let-7; breast cancer; metastasis; drug resistance Received : November 09, 2016 Accepted : January 11, 2017 Published : January 29, 2017 Abstract The RNA binding protein Lin28 is best known for the critical role in cell development, recent researches also have implied its oncogenic function in various human cancers, including breast cancer. Specifically, aberrant Lin28 participates in multiple pathological processes, such as proliferation, metastasis, radiotherapy and chemotherapy resistance, metabolism, immunity and inflammation as well as stemness. In this review, we summarize the let-7-dependent and let-7-independent mechanism regulated by Lin28, focusing on its relation with tumor hallmarks in breast cancer, and subsequently discuss our present knowledge of Lin28 to develop a molecular-based therapeutic strategy against breast cancer.

Background Tamoxifen resistance remains a clinical challenge for hormone receptor-positive breast cancer. Recently, dysregulations in autophagy have been suggested as a potential mechanism for tamoxifen resistance. Although the long noncoding RNA H19 is involved in various stages of tumorigenesis, its role in tamoxifen resistance remains unknown. Here, we assessed the role of H19 in the development of tamoxifen-resistant breast cancer. Methods Quantitative real-time PCR analyzed expression of H19 in tamoxifen-resistant breast cancer tissues. Knockdown of H19 was used to assess the sensitivity to tamoxifen in vitro and in vivo. Both knockdown and overexpression of H19 were used to analyze the status of autophagy. Real-time quantitative methylation-specific polymerase chain reaction, chromatin immunoprecipitation, immunofluorescence, and Western blot were used to explore the tamoxifen resistance mechanism of H19. Results In this study, we observed that the expression of H19 was substantially upregulated in tamoxifen-resistant breast cancer cell line and tumor tissues, and knockdown of H19 enhanced the sensitivity to tamoxifen both in vitro and in vivo. Furthermore, knockdown of H19 significantly inhibited autophagy in MCF7 tamoxifen-resistant (MCF7/TAMR) cells. Conversely, overexpression of H19 promoted autophagy. Interestingly, overexpression of H19 in MCF7 tamoxifen-sensitive cells could recapitulate tamoxifen resistance. Moreover, an increase in methylation in the promoter region of Beclin1 was observed in MCF7/TAMR-shH19 cells. In the double knockdown groups, both shH19+shSAHH and shH19+shDNMT3B rescued the Beclin1 promoter region methylation levels and reactivated autophagy functions. A chromatin immunoprecipitation assay further validated that DNMT3B binds to the Beclin1 promoter region and the knockdown of H19 increases this binding. Conclusions Our findings demonstrate that H19 induces autophagy activation via the H19/SAHH/DNMT3B axis, which could contribute to tamoxifen resistance in breast cancer. Electronic supplementary material The online version of this article (10.1186/s13045-019-0747-0) contains supplementary material, which is available to authorized users.

Cong Chen,1,2 Shumin Huang,3 Aihua Huang,2,4 Yunlu Jia,1,2 Ji Wang,1,2 Zeqin Zhang,1,2 Misha Mao,1,2 Linbo Wang,1,2 Jichun Zhou1,2 1Department of Surgical Oncology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China; 2Biomedical Research Center and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China; 3The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, China; 4Department of Pathology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China Background: Ductal carcinoma in situ with microinvasion (DCISM) represents ~1% of all breast cancer cases. Risk factors for lymph node (LN) metastasis and appropriate adjuvant therapy for DCISM are still widely debated. Methods: We retrieved DCISM data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results registry database (1998–2013). Chi-squared tests and logistic regression models were applied to investigate the potential risks of LN metastasis. Univariate and multivariate Cox proportional hazards regressions were performed to estimate the prognostic factors of DCISM. Survival outcomes were estimated using the Kaplan–Meier method. A 1:1 propensity score matching was used to minimize potential bias. Results: Overall, 6,219 patients with DCISM met our inclusion criteria. Younger age and higher grade disease were identified as risk factors for LN metastasis. In the multivariable analysis, LN metastasis and chemotherapy were prognostic factors for worse overall survival and breast cancer-specific survival. Furthermore, propensity score matching and subgroup analysis showed that chemotherapy may not be effective for DCISM patients. Conclusion: Younger patients with high-grade disease tend to have LN involved in DCISM. Adjuvant chemotherapy might not be necessary for patients with DCISM. Keywords: SEER database, breast cancer, ductal carcinoma in situ with microinvasion, adjuvant chemotherapy, lymphatic metastasis

Cyclooxygenase-2 (COX-2) plays an important role in lung cancer development and progression. Using streptavidin-agarose pulldown and proteomics assay, we identified and validated Ku80, a dimer of Ku participating in the repair of broken DNA double strands, as a new binding protein of the COX-2 gene promoter. Overexpression of Ku80 up-regulated COX-2 promoter activation and COX-2 expression in lung cancer cells. Silencing of Ku80 by siRNA down-regulated COX-2 expression and inhibited tumor cell growth in vitro and in a xenograft mouse model. Ku80 knockdown suppressed phosphorylation of ERK, resulting in an inactivation of the MAPK pathway. Moreover, CBP, a transcription co-activator, interacted with and acetylated Ku80 to co-regulate the activation of COX-2 promoter. Overexpression of CBP increased Ku80 acetylation, thereby promoting COX-2 expression and cell growth. Suppression of CBP by a CBP-specific inhibitor or siRNA inhibited COX-2 expression as well as tumor cell growth. Tissue microarray immunohistochemical analysis of lung adenocarcinomas revealed a strong positive correlation between levels of Ku80 and COX-2 and clinicopathologic variables. Overexpression of Ku80 was associated with poor prognosis in patients with lung cancers. We conclude that Ku80 promotes COX-2 expression and tumor growth and is a potential therapeutic target in lung cancer.

// Wenxian Hu 1, * , Yunlu Jia 1, * , Xiangsheng Xiao 2 , Kezhen Lv 3 , Yongxia Chen 1 , Linbo Wang 1 , Xiao Luo 1 , Tianze Liu 2 , Wenbin Li 2 , Yixin Li 2 , Changlin Zhang 2 , Zhenglong Yu 4 , Wenlin Huang 2, 5 , Bing Sun 4 , Wu-guo Deng 2, 5 1 Department of Surgical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China 2 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China 3 Department of Breast Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China 4 Department of Thoracic Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian, China 5 State Key Laboratory of Targeted Drug for Tumors of Guangdong Province, Guangzhou Double Bioproduct Inc., Guangzhou, China * These authors contributed equally to this work Correspondence to: Wenxian Hu, email: 13967169904@163.com Bing Sun, email: dysunbing@163.com Wu-guo Deng, email: dengwg@sysucc.org.cn Keywords: KLF4, hTERT, MAPK, lung cancer, cancer prognosis Received: January 07, 2016 Accepted: April 16, 2016 Published: May 02, 2016 ABSTRACT Kruppel-like factor 4 (KLF4) is a transcription factor that contributes to diverse cellular processes and serves as a tumor suppressor or oncogene in various cancers. Previously, we have reported on the tumor suppressive function of KLF4 in lung cancer; however, its precise regulatory mechanism remains elusive. In this study, we found that KLF4 negatively regulated hTERT expression and telomerase activity in lung cancer cell lines and a mouse model. In addition, the KLF4 and hTERT expression levels were significantly related to the clinicopathological features of lung cancer patients. Promoter reporter analyses revealed the decreased hTERT promoter activity in cells infected with Ad-KLF4, and chromatin immunoprecipitation analysis demonstrated that endogenous KLF4 directly bound to the promoter region of hTERT. Furthermore, the MAPK signaling pathway was revealed to be involved in the KLF4/hTERT modulation pathway. Forced expression of KLF4 profoundly attenuated lung cell proliferation and cancer formation in a murine model. Moreover, hTERT overexpression can partially rescue the KLF4-mediated suppressive effect in lung cancer cells. Taken together, these results demonstrate that KLF4 suppresses lung cancer growth by inhibiting hTERT and MAPK signaling. Additionally, the KLF4/hTERT/MAPK pathway is a potential new therapeutic target for human lung cancer.

Krüppel-like factor 4 (KLF4) has critical roles in breast cancer development and progression and several solid tumors. Tamoxifen (TAM) resistance represents a daunting challenge to the successful treatment of breast cancer. KLF4 expression, function, and regulation in the efficacy of TAM therapy in breast cancer have yet to be demonstrated. Here, we investigated the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 was correlated with increased TAM sensitivity in breast cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERa and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness of MCF-7/TAM and T47D cells to TAM. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion, and migration ability. Besides, KLF4 expression was downregulated in breast cancer tumor tissues and high expression of KLF4 linked to favorable outcome. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and P38 were relatively activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors can significantly suppress cell viability. Knockdown of KLF4 activated ERK and P38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and P38 signaling and resulted in enhanced sensitivity to TAM. Therefore, our findings suggest that KLF4 contributes to TAM efficiency in breast cancer via phosphorylation modification of ERK and P38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting the KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for TAM-resistant patients. Note: This abstract was not presented at the conference. Citation Format: Yunlu Jia, Wang Linbo. KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A46.