Chang Gong

let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells

Cancers may arise from rare self-renewing tumor-initiating cells (T-IC). However, how T-IC self renewal, multipotent differentiation, and tumorigenicity are maintained remains obscure. Because miRNAs can regulate cell-fate decisions, we compared miRNA expression in self-renewing and differentiated cells from breast cancer lines and in breast T-IC (BT-IC) and non-BT-IC from 1 degrees breast cancers. let-7 miRNAs were markedly reduced in BT-IC and increased with differentiation. Infecting BT-IC with let-7-lentivirus reduced proliferation, mammosphere formation, and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice, while antagonizing let-7 by antisense oligonucleotides enhanced in vitro self renewal of non-T-IC. Increased let-7 paralleled reduced H-RAS and HMGA2, known let-7 targets. Silencing H-RAS in a BT-IC-enriched cell line reduced self renewal but had no effect on differentiation, while silencing HMGA2 enhanced differentiation but did not affect self renewal. Therefore let-7 regulates multiple BT-IC stem cell-like properties by silencing more than one target.

Up-regulation of miR-21 Mediates Resistance to Trastuzumab Therapy for Breast Cancer

Trastuzumab resistance emerges to be a major issue in anti-human epidermal growth factor receptor 2 (HER2) therapy for breast cancers. Here, we demonstrated that miR-21 expression was up-regulated and its function was elevated in HER2+ BT474, SKBR3, and MDA-MB-453 breast cancer cells that are induced to acquire trastuzumab resistance by long-term exposure to the antibody, whereas protein expression of the PTEN gene, a miR-21 target, was reduced. Blocking the action of miR-21 with antisense oligonucleotides re-sensitized the resistant cells to the therapeutic activities of trastuzumab by inducing growth arrest, proliferation inhibition, and G1-S cell cycle checking in the presence of the antibody. Ectopic expression of miR-21 in HER2+ breast cancer cells confers resistance to trastuzumab. Rescuing PTEN expression with a p3XFLAG-PTEN-mut construct with deleted miR-21 targeting sequence at its 3′ UTR restored the growth inhibition of trastuzumab in the resistant cells by inducing PTEN activation and AKT inhibition. In vivo, administering miR-21 antisense oligonucleotides restored trastuzumab sensitivity in the resistant breast cancer xenografts by inducing PTEN expression, whereas injection of miR-21 mimics conferred trastuzumab resistant in the sensitive breast tumors via PTEN silence. Up-regulatin of miR-21 in tumor biopsies obtained from patients receiving pre-operative trastuzumab therapy was associated with poor trastuzumab response. Therefore, miR-21 overexpression contributes to trastuzumab resistance in HER2+ breast cancers and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to anti-HER2 treatment.

Long non-coding RNA HOTAIR is an independent prognostic marker for nasopharyngeal carcinoma progression and survival

Identification of new nasopharyngeal carcinoma (NPC) biomarkers is of great clinical value for the diagnosis and treatment of NPC. HOTAIR, a cancer‐related long non‐coding RNA, was tested and its prognostic value for NPC was evaluated. As determined using in situ hybridization (ISH), 91 of 160 (56.87%) paraffin‐embedded NPC biopsies showed high expression levels of HOTAIR (staining index score ≥ 6). HOTAIR was upregulated in tumors with a large size (P = 0.021), more advanced clinical stage (P = 0.012) and increased lymph node tumor burden (P = 0.005). Quantified using real‐time PCR, HOTAIR expression levels in fresh tissue and paraffin‐embedded samples were 5.2 ~ 48.4‐fold higher compared with non‐cancer tissue samples. Moreover, HOTAIR expression levels increased with clinical stage progression, which was consistent with ISH findings in the paraffin‐embedded tissue. Most importantly, NPC patients with higher HOTAIR levels had a poor prognosis for overall survival using univariate and multivariate analysis. In addition, HOTAIR mediated the migration, invasion and proliferation of NPC cells in vitro. HOTAIR is a potential biomarker for the prognosis of NPC, and dysregulation of HOTAIR might play an important role in NPC progression.

Reduced miR-128 in breast tumor-initiating cells induces chemotherapeutic resistance via Bmi-1 and ABCC5

Purpose: Tumor-initiating cells are resistant to chemotherapy, but how microRNAs play a role in regulating drug resistance of breast tumor–initiating cells (BT-IC) needs to be clarified. Experimental Design: Lentivirus-mediated miR-128 transduction was done in BT-ICs, enriched by mammosphere cultures or CD44+CD24− fluorescence-activated cell sorting. Apoptosis and DNA damage were determined upon treatment with doxorubicin. Expression of miR-128 in breast cancer tissues was examined by in situ hybridization and correlated with breast tumor response to neoadjuvant chemotherapy and patient survival. Results: MiR-128 was significantly reduced in chemoresistant BT-ICs enriched from breast cancer cell lines and primary breast tumors (P < 0.01), accompanied by an overexpression of Bmi-1 and ABCC5, which were identified as targets of miR-128. Ectopic expression of miR-128 reduced the protein levels of Bmi-1 and ABCC5 in BT-ICs, along with decreased cell viability (P < 0.001) and increased apoptosis (P < 0.001) and DNA damage (P < 0.001) in the presence of doxorubicin. Reduced miR-128 expression in breast tumor tissues was associated with chemotherapeutic resistance (P < 0.001) and poor survival of breast cancer patients (P < 0.05; n = 57). Conclusions: Reduction in miR-128 leading to Bmi-1 and ABCC5 overexpression is a stem cell–like feature of BT-ICs, which contributes to chemotherapeutic resistance in breast cancers. Ectopic expression of miR-128 sensitizes BT-ICs to the proapoptotic and DNA-damaging effects of doxorubicin, indicating therapeutic potential. Clin Cancer Res; 17(22); 7105–15. ©2011 AACR.

Glutathion s transferase π indicates chemotherapy resistance in breast cancer

BACKGROUND Breast cancer is the most common malignant disease of women. Pathologic response of breast cancer to chemotherapy has a great prognostic importance. Glutathion S Transferases (GSTs) might detoxify chemotherapeutic drugs within the cancer cells, thus contributing to chemotherapy resistance. The pi isoenzyme of GSTs seems to be of great relevance. Thus, we hypothesized that GSTpi expression in cancer biopsy can be a prognostic indicator for resistance to chemotherapy. To test this hypothesis, we evaluated before and after chemotherapy, tumor size, apoptosis of tumor cells with TUNEL assay, and proliferation of tumor cells by determining PCNA expression in biopsy samples, or in the surgically removed tumor tissue of GSTpi (-), and GSTpi (+) cases. MATERIALS AND METHODS GSTpi immunoreactivity was determined in 42 female patients with breast cancer. Patients were divided into two groups according to the expression of GSTpi in the pre-treatment biopsy specimen: (+) (n = 22) and (n = 20) samples were analyzed. Surgery was performed 2 weeks after a single intravenous injection of the chemotherapeutic drugs [5-fluorouracil, adriamycin, mitomycin (FAM protocol)]. RESULTS Pre-chemotherapy values of tumor size, apoptosis, or proliferation did not differ between GSTpi (-) and (+) samples. Chemotherapy significantly inhibited tumor growth, and cell proliferation, and induced apoptosis in GSTpi (-) cases. However, these effects were significantly reduced in GSTpi (+) patients. CONCLUSION These results suggest, that the presence of GSTpi in breast cancer tissue is a bad prognostic indicator, and these tumors are largely resistant to chemotherapy. Thus, GSTpi might be important in inactivating one or more of the chemotherapeutic agents used in this treatment.

Markers of tumor-initiating cells predict chemoresistance in breast cancer.

Purpose Evidence is lacking whether the number of breast tumor-initiating cells (BT-ICs) directly correlates with the sensitivity of breast tumors to chemotherapy. Here, we evaluated the association between proportion of BT-ICs and chemoresistance of the tumors. Methods Immunohistochemical staining(IHC) was used to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and proliferating cell nuclear antigen, and TUNEL was used to detect the apoptosis index. The significance of various variables in patient survival was analyzed using a Cox proportional hazards model. The percentage of BT-ICs in breast cancer cell lines and primary breast tumors was determined by ALDH1 enzymatic assay, CD44+/CD24− phenotype and mammosphere formation assay. Results ALDH1 expression determined by IHC in primary breast cancers was associated with poor clinical response to neoadjuvant chemotherapy and reduced survival in breast cancer patients. Breast tumors that contained higher proportion of BT-ICs with CD44+/CD24− phenotype, ALDH1 enzymatic activity and sphere forming capacity were more resistant to neoadjuvant chemotherapy. Chemoresistant cell lines AdrR/MCF-7 and SK-3rd, had increased number of cells with sphere forming capacity, CD44+/CD24− phenotype and side-population. Regardless the proportion of T-ICs, FACS-sorted CD44+/CD24− cells that derived from primary tumors or breast cancer lines were about 10–60 fold more resistant to chemotherapy relative to the non- CD44+/CD24− cells and their parental cells. Furthermore, our data demonstrated that MDR1 (multidrug resistance 1) and ABCG2 (ATP-binding cassette sub-family G member 2) were upregulated in CD44+/CD24− cells. Treatment with lapatinib or salinomycin reduced the proportion of BT-ICs by nearly 50 fold, and thus enhanced the sensitivity of breast cancer cells to chemotherapy by around 30 fold. Conclusions These data suggest that the proportion of BT-ICs is associated with chemotherapeutic resistance of breast cancer. It highlights the importance of targeting T-ICs, rather than eliminating the bulk of rapidly dividing and terminally differentiated cells, in novel anti-cancer strategies.

Plasma miR-221 as a Predictive Biomarker for Chemoresistance in Breast Cancer Patients who Previously Received Neoadjuvant Chemotherapy

Background: Neoadjuvant chemotherapy (NAC) is increasingly being used in breast cancer treatment. Research has revealed an elevated expression of miR-221 in adriamycinresistant MCF-7/ADR cells. This study aimed to explore the potential role of miR-221 as a biomarker for chemosensitivity in breast cancer patients who previously received NAC. Patients and Methods: The expression levels of circulating miR-221 in the plasma of 93 breast cancer patients who previously received NAC and in 32 healthy individuals were assessed. The correlations between miR-221 and clinicopathological features and chemosensitivity were also analysed. Results: The expression level of miR-221 was significantly associated with hormone receptor (HR) status (p = 0.008). Patients with higher plasma miR-221 levels tended to be HR-negative. Patients with different miR-221 levels had significant differences in the overall response rate (p = 0.044) but not in the pathologic complete response rate (p = 0.477). Conclusion: Our results indicate that plasma miR-221 may be a predictive biomarker for sensitivity to NAC in breast cancer patients.

miR-21 Induces Myofibroblast Differentiation and Promotes the Malignant Progression of Breast Phyllodes Tumors

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell-derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA-positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21.

MiR-106b expression determines the proliferation paradox of TGF-β in breast cancer cells

TGF-β has paradoxical effects on cancer cell proliferation, as it suppresses proliferation of normal epithelial and low-invasive cancer cells, but enhances that of high-invasive cancer cells. However, how cancer cells acquire the ability to evade the tumor-suppressing effects of TGF-β, yet still take advantage of its tumor-promoting effects, remains elusive. Here, we identified miR-106b as a molecular switch to determine TGF-β effects on cell proliferation. TGF-β1 enhances the transcription of miR-106b via a promoter independent of its host gene MCM7 by activating c-jun. In high-invasive breast cancer cells, miR-106b is upregulated by TGF-β1 at a much higher level than that in normal or low-invasive cancer cells. Accumulation of miR-106b counterbalances TGF-β growth-inhibiting effects by eliminating activated retinoblastoma (RB) and results in enhanced proliferation. Furthermore, miR-106b mediates TGF-β effects on tumor growth and metastasis in breast cancer xenografts. In addition, miR-106b expression is elevated in higher stage tumors and correlated with tumor progression in breast cancer patients. These findings suggest that high level of miR-106b induced by TGF-β determines the tumor-promoting effects of TGF-β in breast cancer.

BRMS1L suppresses breast cancer metastasis by inducing epigenetic silence of FZD10.

BRMS1L (breast cancer metastasis suppressor 1 like, BRMS1-like) is a component of Sin3A-histone deacetylase (HDAC) co-repressor complex that suppresses target gene transcription. Here we show that reduced BRMS1L in breast cancer tissues is associated with metastasis and poor patient survival. Functionally, BRMS1L inhibits breast cancer cells migration and invasion by inhibiting epithelial-mesenchymal transition. These effects are mediated by epigenetic silencing of FZD10, a receptor for Wnt signalling, through HDAC1 recruitment and histone H3K9 deacetylation at the promoter. Consequently, BRMS1L-induced FZD10 silencing inhibits aberrant activation of WNT3-FZD10-β-catenin signalling. Furthermore, BRMS1L is a target of miR-106b and miR-106b upregulation leads to BRMS1L reduction in breast cancer cells. RNA interference-mediated silencing of BRMS1L expression promotes metastasis of breast cancer xenografts in immunocompromised mice, whereas ectopic BRMS1L expression inhibits metastasis. Therefore, BRMS1L provides an epigenetic regulation of Wnt signalling in breast cancer cells and acts as a breast cancer metastasis suppressor.