ZFP64 drives glycolysis-mediated stem cell-like properties and tumorigenesis in breast cancer.

BACKGROUND: Breast cancer (BC) is a great clinical challenge because of its aggressiveness and poor prognosis. Zinc Finger Protein 64 (ZFP64), as a transcriptional factor, is responsible for the development and progression of cancers. This study aims to investigate whether ZFP64 regulates stem cell-like properties and tumorigenesis in BC by the glycolytic pathway. RESULTS: It was demonstrated that ZFP64 was overexpressed in BC specimens compared to adjacent normal tissues, and patients with high ZFP64 expression had shorter overall survival and disease-free survival. The analysis of the association of ZFP64 expression with clinicopathological characteristics showed that high ZFP64 expression is closely associated with N stage, TNM stage, and progesterone receptor status. Knockdown of ZFP64 suppressed the viability and colony formation capacity of BC cells by CCK8 and colony formation assays. The subcutaneous xenograft models revealed that ZFP64 knockdown reduced the volume of formatted tumors, and decreased Ki67 expression in tumors. The opposite effects on cell proliferation and tumorigenesis were demonstrated by ZFP64 overexpression. Furthermore, we suggested that the stem cell-like properties of BC cells were inhibited by ZFP64 depletion, as evidenced by the decreased size and number of formatted mammospheres, the downregulated expressions of OCT4, Nanog, and SOX2 proteins, as well as the reduced proportion of CD44+/CD24- subpopulations. Mechanistically, glycolysis was revealed to mediate the effect of ZFP64 using mRNA-seq analysis. Results showed that ZFP64 knockdown blocked the glycolytic process, as indicated by decreasing glycolytic metabolites, inhibiting glucose consumption, and reducing lactate and ATP production. As a transcription factor, we identified that ZFP64 was directly bound to the promoters of glycolysis-related genes (ALDOC, ENO2, HK2, and SPAG4), and induced the transcription of these genes by ChIP and dual-luciferase reporter assays. Blocking the glycolytic pathway by the inhibition of glycolytic enzymes ENO2/HK2 suppressed the high proliferation and stem cell-like properties of BC cells induced by ZFP64 overexpression. CONCLUSIONS: These data support that ZFP64 promotes stem cell-like properties and tumorigenesis of BC by activating glycolysis in a transcriptional mechanism.

Cell adhesion molecule CD44v10 promotes stem-like properties in triple-negative breast cancer cells via glucose transporter GLUT1-mediated glycolysis.

Cell adhesion molecule CD44v8-10 is associated with tumor ste0mness and malignancy; however, whether CD44v10 alone confers these properties is unknown. Here, we demonstrated that CD44v10 promotes stemness and chemoresistance of triple-negative breast cancers (TNBCs) individually. Next, we identified that genes differentially expressed in response to ectopic expression of CD44v10 are mostly related to glycolysis. Further, we showed that CD44v10 upregulates glucose transporter 1 to facilitate glycolysis by activating the MAPK/ERK and PI3K/AKT signaling pathways. This glycolytic reprogramming induced by CD44v10 contributes to the stem-like properties of TNBC cells and confers resistance to paclitaxel treatment. Notably, we determined that the knockdown of glucose transporter 1 could attenuate the enhanced effects of CD44v10 on glycolysis, stemness, and paclitaxel resistance. Collectively, our findings provide novel insights into the function of CD44v10 in TNBCs and suggest that targeting CD44v10 may contribute to future clinical therapy.

LncRNA DUXAP10 Upregulation and the Hedgehog Pathway Activation Are Critically Involved in Chronic Cadmium Exposure-Induced Cancer Stem Cell-Like Property.

Cadmium (Cd) is a well-known lung carcinogen. However, the mechanism of Cd carcinogenesis remains to be clearly defined. Cd has been shown to act as a weak mutagen, suggesting that it may exert tumorigenic effect through nongenotoxic ways, such as epigenetic mechanisms. Long noncoding RNAs (lncRNAs) refer to RNA molecules that are longer than 200 nucleotides in length but lack protein-coding capacities. Regulation of gene expressions by lncRNAs is considered as one of important epigenetic mechanisms. The goal of this study is to investigate the mechanism of Cd carcinogenesis focusing on the role of lncRNA dysregulations. Cd-induced malignant transformation of human bronchial epithelia BEAS-2B cells was accomplished by a 9-month low-dose Cd (CdCl2, 2.5 µM) exposure. The Cd-exposed cells formed significantly more colonies in soft agar, displayed cancer stem cell (CSC)-like property, and formed tumors in nude mice. Mechanistically, chronic low-dose Cd exposure did not cause significant genotoxic effects but dysregulated lncRNA expressions. Further Q-PCR analysis confirmed the significant upregulation of the oncogenic lncRNA DUXAP10 in Cd-transformed cells. DUXAP10 knockdown in Cd-transformed cells significantly reduced their CSC-like property. Further mechanistic studies showed that the Hedgehog pathway is activated in Cd-transformed cells and inhibition of this pathway reduces Cd-induced CSC-like property. DUXAP10 knockdown caused the Hedgehog pathway inactivation in Cd-transformed cells. Furthermore, Pax6 expression was upregulated in Cd-transformed cells and Pax6 knockdown significantly reduced their DUXAP10 levels and CSC-like property. In summary, these findings suggest that the lncRNA DUXAP10 upregulation may play an important role in Cd carcinogenesis.

LGR5 induces ß-catenin activation and augments tumour progression by activating STAT3 in human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: LGR5 enhances Wnt-ß-catenin signalling; however, involvement of LGR5 or Wnt-ß-catenin signalling in ICC progression has not been reported. METHODS: Functions and regulations of LGR5-mediated ß-catenin activation in ICC progression were evaluated using surgical specimens collected from 61 ICC patients or 2 ICC cell lines. RESULTS: LGR5 expression was increased in some cases of ICC. It was positively correlated with ß-catenin activation, OLFM4 expression and STAT3 activation, and negatively correlated with GRIM19 expression in ICC, thereby enhancing cancer stem cell (CSC)-like property and EMT. High LGR5 expression was an independent factor for poor prognosis in ICC after operation. In vitro, Wnt inhibition by IWP-2 suppressed ß-catenin activation, OLFM4 expression and STAT3 activation. IWP-2 treatment decreased expression of EpCAM, CD133, vimentin and increased E-cadherin expression. The rate of mesenchymal cells was decreased and cell invasiveness was suppressed after IWP-2 treatment, suggesting that Wnt-ß-catenin signalling enhanced CSC-like property and EMT by activating STAT3. In addition, LGR5 knockdown inhibited ß-catenin activation, resulting in suppression of ß-catenin-induced STAT3 activation through inhibition of OLFM4-GRIM19 cascade. As these results, LGR5 knockdown suppressed CSC-like property and EMT. Therefore, LGR5 was a key regulator for ß-catenin activation, and ß-catenin was unable to be activated without LGR5. CONCLUSIONS: LGR5 is essential for ß-catenin activation induced by Wnt signalling. Activated ß-catenin further activates STAT3 and enhances CSC-like property and EMT, leading to aggressive tumour progression and poor prognosis in patients with ICC. Therefore, LGR5 is an excellent prognostic predictor and a promising therapeutic target for ICC.

Deubiquitinase USP7-mediated MCL-1 up-regulation enhances Arsenic and Benzo(a)pyrene co-exposure-induced Cancer Stem Cell-like property and Tumorigenesis.

Rationale: MCL-1 is up-regulated in cancer and a target for cancer treatment. How MCL-1 is up-regulated and whether MCL-1 up-regulation plays a role in tumorigenic process is not well-known. Arsenic and benzo(a)pyrene (BaP) are well-recognized lung carcinogens and we recently reported that arsenic and BaP co-exposure acts synergistically in inducing cancer stem cell (CSC)-like property and lung tumorigenesis. This study was performed to further investigate the underlying mechanism focusing on the role of MCL-1. Methods: The spheroid formation assay and nude mouse tumorigenesis assay were used to determine the CSC-like property and tumorigenicity of arsenic plus BaP co-exposure-transformed human bronchial epithelial BEAS-2B cells, respectively. Biochemical, pharmacological and genetic approaches were used to manipulate gene expressions, dissect signaling pathways and determine protein-protein interactions. Both loss-of-function and gain-of-function approaches were used to validate the role of MCL-1 in arsenic plus BaP co-exposure-enhanced CSC-like property and tumorigenicity. Results: Arsenic plus BaP co-exposure-transformed cells express significantly higher protein levels of MCL-1 than the passage-matched control, arsenic or BaP exposure alone-transformed cells. Knocking down MCL-1 levels in arsenic plus BaP co-exposure-transformed cells significantly reduced their apoptosis resistance, CSC-like property and tumorigenicity in mice. Mechanistic studies revealed that arsenic plus BaP co-exposure up-regulates MCL-1 protein levels by synergistically activating the PI3K/Akt/mTOR pathway to increase the level of a deubiquitinase USP7, which in turn reduces the level of MCL-1 protein ubiquitination and prevents its subsequent proteasome degradation. Conclusions: The deubiquitinase USP7-mediated MCL-1 up-regulation enhances arsenic and BaP co-exposure-induced CSC-like property and tumorigenesis, providing the first evidence demonstrating that USP7 stabilizes MCL-1 protein during the tumorigenic process.

A Positive Feedback Loop Between c-Myc Upregulation, Glycolytic Shift, and Histone Acetylation Enhances Cancer Stem Cell-like Property and Tumorigenicity of Cr(VI)-transformed Cells.

Chronic hexavalent chromium [Cr(VI)] exposure causes lung cancer and other types of cancer; however, the mechanism of Cr(VI) carcinogenesis remains to be clearly defined. Our recent study showed that chronic Cr(VI) exposure upregulates the proto oncogene c-Myc expression, which contributes significantly to Cr(VI)-induced cell transformation, cancer stem cell (CSC)-like property and tumorigenesis. c-Myc is a master regulator of cancer cell abnormal metabolism and accumulating evidence suggests that metabolism dysregulation plays an important role in both cancer development and progression. However, little is known about the role of metabolism dysregulation in Cr(VI) carcinogenesis. This study was performed to investigate the potential role and mechanism of metabolism dysregulation in Cr(VI) carcinogenesis. It was found that Cr(VI)-transformed cells display glycolytic shift, which depends on the upregulation of c-Myc. The glycolytic shift in Cr(VI)-transformed cells led to increased production of acetyl coenzyme A (acetyl-CoA) and elevation of histone acetylation. This, in turn, upregulated the expression of an acetyl-CoA producing key enzyme ATP citrate lyase and c-Myc, forming a positive feedback loop between the upregulation of c-Myc expression, glycolytic shift and increased histone acetylation. It was further determined that glucose depletion not only reverses the glycolytic shift in Cr(VI)-transformed cells, but also significantly reduces their growth, CSC-like property and tumorigenicity. These findings indicate that glycolytic shift plays an important role in maintaining malignant phenotypes of Cr(VI)-transformed cells, suggesting that metabolism dysregulation is critically involved in Cr(VI) carcinogenesis.

miR-326 inhibits the cell proliferation and cancer stem cell-like property of cervical cancer in vitro and oncogenesis in vivo via targeting TCF4.

BACKGROUND: Cervical cancer ranks as one of the most prevalent female malignancies globally, and its treatment with new targets has been the focus of current research. The present study set out to investigate the function of microRNA-326 (miR-326) in vitro and in vivo and to verify the direct targeting of transcription factor 4 (TCF4) by miR-326. METHODS: The detection of messenger RNA (mRNA) expressing miR-326 and TCF4 in cervical cancer cell lines and tumor samples was conducted using quantitative real-time polymerase chain (qRT-PCR). A dual-luciferase reporter assay was carried out to detect the target relationship of miR-326 with TCF4. A Cell Counting Kit-8 (CCK-8) assay was employed to detect the effect of miR-326 on CasKi cell viability. Flow cytometry and western blotting were employed to examine the effects of miR-326 on cancer stem cell (CSC)-like property. Tumor weight was measured in orthotopic xenograft mouse models. Immunohistochemistry was employed to analyze the protein expression levels of Ki-67, proliferating cell nuclear antigen (PCNA), CD44, and SRY-box 4 (SOX4). RESULT: Downregulation of the mRNA expression levels of miR-326 was observed in cervical cancer cell lines and tumor tissue, while the levels of TCF4 were upregulated. The dual-luciferase reporter assay revealed binding of miR-326 to the three prime untranslated region (3'-UTR) of TCF4. In vitro assays demonstrated that miR-326 inhibited CasKi cell proliferation through regulating TCF4. miR-326 also suppressed the CSC-like property of CasKi cells by targeting TCF4. Furthermore, the protein expression levels of cyclin D1, ß-catenin, and c-Myc were decreased when miR-326 was added to TCF4-transfected cells. In vivo assays demonstrated that miR-326 inhibited tumor weight, growth, and the protein expression levels of Ki-67, PCNA, CD44, SOX4, and ß-catenin. CONCLUSIONS: miR-326 acted in a tumor-suppressive manner through its regulation of TCF4, and has potential as a biomarker or therapeutic target for cervical cancer.

Chronic Hexavalent Chromium Exposure Induces Cancer Stem Cell-Like Property and Tumorigenesis by Increasing c-Myc Expression.

Hexavalent chromium [Cr(VI)] is one of the most common environmental carcinogen causing lung cancer in humans; however, the mechanism of Cr(VI) carcinogenesis remains elusive. Cancer stem cells (CSCs) are considered as cancer initiating and maintaining cells. Ours and other recent studies showed that chronic Cr(VI) exposure induces CSC-like property representing an important mechanism of Cr(VI) carcinogenesis. However, how Cr(VI) exposure induces CSC-like property remains largely unknown. In this study, we found that stably knocking down the expression of c-Myc, a proto-oncogene and one of key stemness factors playing critical roles in cancer initiation and progression, in Cr(VI)-transformed human bronchial epithelial cells [BEAS-2B-Cr(VI)] significantly decreased their CSC-like property and tumorigenicity in mice. Moreover, stably knocking down c-Myc expression in parental nontransformed BEAS-2B cells significantly impaired the capability of chronic Cr(VI) exposure to induce CSC-like property and cell transformation. It was also found that stably overexpressing c-Myc alone in parental nontransformed BEAS-2B cells is capable of causing CSC-like property and cell transformation. Mechanistic studies showed that chronic Cr(VI) exposure increases c-Myc expression by down-regulating the level of microRNA-494 (miR-494). It was further determined that overexpressing miR-494 significantly reduces Cr(VI)-induced CSC-like property, cell transformation, and tumorigenesis mainly through down-regulating c-Myc expression. Together, these findings indicate that chronic low dose Cr(VI) exposure induces CSC-like property and tumorigenesis by increasing c-Myc expression through down-regulating the level of miR-494, revealing an important role of the proto-oncogene c-Myc in Cr(VI) carcinogenesis.

The metastasis suppressor, NDRG1, inhibits "stemness" of colorectal cancer via down-regulation of nuclear ß-catenin and CD44.

N-myc downstream-regulated gene 1 (NDRG1), has been identified as an important metastasis suppressor for colorectal cancer (CRC). In this study, we investigated: (1) the effects of NDRG1 on CRC stemness and tumorigenesis; (2) the molecular mechanisms involved; and (3) the relationship between NDRG1 expression and colorectal cancer prognosis. Our investigation demonstrated that CRC cells with silenced NDRG1 showed more tumorigenic ability and stem cell-like properties, such as: colony and sphere formation, chemoresistance, cell invasion, high expression of CD44, and tumorigenicity in vivo. Moreover, NDRG1 silencing reduced ß-catenin expression on the cell membrane, while increasing its nuclear expression. The anti-tumor activity of NDRG1 was demonstrated to be mediated by preventing ß-catenin nuclear translocation, as silencing of this latter molecule could reverse the effects of silencing NDRG1 expression. NDRG1 expression was also demonstrated to be negatively correlated to CRC prognosis. In addition, there was a negative correlation between NDRG1 and nuclear ß-catenin and also NDRG1 and CD44 expression in clinical CRC specimens. Taken together, our investigation demonstrates that the anti-metastatic activity of NDRG1 in CRC occurs through the down-regulation of nuclear ß-catenin and suggests that NDRG1 is a significant therapeutic target.