| Tumor recurrence accompanied by resistance to treatment, i.e., the tumors respond to initial therapies but eventually grow back and become resistant to chemo/radiotherapy, remains an unmet medical need. Recent studies suggested that the drug-resistance of recurred tumor may be due to a special small side-population in the tumor, named tumor-initiating cells or cancer stem cells. Cancer stem cells are only a small fraction in tumor, capable of self-renewal, unlimited growth and differentiation, are responsible for tumor outgrowth, progression, drug-resistance and metastasis.More recent studies demonstrated that microRNAs (miRNAs) were involved in the regulation of the self-renewal and difference of cancer stem cells. MicroRNAs are a conserved class of non-coding 20-22nt small RNAs that regulate gene expression by binding to mRNA leading to mRNA degradation or inhibition. It has been shown that certain miRNAs are involved in carcinogenesis via regulating cell proliferation and/or cell death, some of which act like oncogenes or tumor suppressor genes.MicroRNA miR-34 was recently found to be a direct target of p53, function at the downstream of p53 pathway as a tumor suppressor. Delineating the role of miR-34 in regulation of cell growth and tumor progression, and its relationship with cancer stem cells, will help us better understand the p53 tumor suppressor signaling network, facilitate our research in carcinogenesis and cancer therapy, and build a solid foundation for our exploration of novel strategies in diagnosis, treatment and prevention of cancer.Our studies demonstrated that miR-34 is involved in p53 tumor suppressor network; restoration of miR-34 is able to re-establish the tumor suppressing signaling pathway in cancer cells lacking functional p53. More significantly, miR-34 can inhibit cancer cell growth and tumor initiation via inhibiting the self-renewal of cancer stem cells, indicated that miR-34 can function as a tumor suppressor gene in p53-deficient cells, and its loss-of-function plays a role in the dysregulation of self-renewal in cancer stem cells.Specific Aim: To investigate the potential role of miR-34 in p53-deficient cancer cells and cancer stem cellsMethods:1. miRNA array analysis in drug-resistant clone CL-1 and its parental cell line LNCaP.Cells from the two cell lines were lyzed to extract total RNA, sent to LC Sciences USA for miRNA array detection. The samples were first labeled with Cy3 or Cy5, respectively, hybridized with MRA-1001 arrays, scanned for fluorescent signal, analyzed by special software for differential expression of miRNAs.2. miR-34 mimics transfection and target genes expressionHuman cancer cells lines were first examined for their basal levels of miR-34 expression by qRT-PCR. miR-34a, miR-34b, miR-34c, and negative control miRNA mimics were synthesized in Dharmacon, transfected into cancer cells with low miR-34, whereas miR-34 antagonists were transfected into cancer cells with high levels of miR-34 expression. Potential target genes expression were assessed by Western blot for proteins, quantitative real-time PCR for mRNAs. miR-34 reporter assay was carried out using Bcl-2-3'UTR reporter construct or its miRNA binding site mutant construct co-transfected with miR-34 mimics, then measured the bioluminescence.3. Establishment and characterization of miR-34 high expression stable clonesThe lentivial miR-34 systems were used to co-infect 293 packaging cells, the virus-containing supernatants were used to infect cancer cells. The stable clones were selected by antibiotic resistance and strong green fluorescence, validated by Real-time PCR for miR-34 expression.4. Effects of miR-34 restoration on human cancer cellsFor the miR-34 mimics transfected cancer cells and miR-34 stable clones, the following assays were employed to examine the biological effects: (1) cells were counted to generate cell growth curves; (2) colony formation assay for clonogenic growth; (3) Cell cycle analysis by flow cytometry; (4) Transwell invasion assay for cell invasion; (5) MTT-based cytotoxicity assay for chemosensitization by miR-34; (6) Caspase-3 activation and sub-G1 analysis for apoptosis induced by miR-34 in combination with chemotherapy and X-ray radiation (chemo/radiosensitization). 5. Effects of miR-34 restoration on cancer stem cellsCancer cells were stained with CD44/CD133 and sorted by FACS, the sorted cells were cultured for tumorspheres formation and growth; or lyzed for total RNA and examined for miR-34 expression.6. Effects of miR-34 restoration on tumor initiation in nude miceThe miR-34 mimics transfected cancer cells and miR-34 stable clones, or the cells from tumorspheres, were inoculated s.q. into NCr-nu/nu athymic nude mice from NCI. The tumors and animal body weight were monitored for tumor formation and growth.Results:1. miRNA array studyChemoresistant p53-deficient CL-1 cells versus sensitive p53wt LNCaP cells, Bcl-2 levels was increased 3-4-fold, became more resistant to chemotherapy, more aggressive and metastatic. miRNA array data showed that CL-1 lost miR-34a expression, LNCaP has 47-fold v.s.CL-1. The data was confirmed by real-time-PCR.2. miR-34 mimics transfection and target genes expressionWestern blot results showed that cancer cells transfected with miR-34 mimics have reduced expression of target genes, Bcl-2, Notch1 and Notch2, consistent with Real-time PCR analysis of their mRNA levels. Bcl-2-3'UTR reporter assay showed that the transfected miR-34 mimics are functional and that Bcl-2 is a direct target of miR-34.3. miR-34 high expression stable clonesLentiviral vector p-miR34a-MIF or vector control p-MIF and lentiviral packaging plasmids pFIV-34N and pVSV-G co-transfected 293 packaging cells, 48 hours later, the lentivirus in supernatant were collected and filtered, used to infect cancer cells with polybreen. After antibiotic Zeocin selection for two weeks, the stable clones were obtained. Real-time PCR confirmed that target genes mRNAs were downregulated; Bcl-2-3'UTR reporter assay showed that miR-34a in the cells are functional.4. Effects of miR-34 restoration on human cancer cellsCompared with control miRNA, cancer cells with restored functional miR-34 have: (1) significantly impaired cell growth; (2) significantly reduced colony formation; (3) G1 block, consistent with p53 restoration; (4) significantly reduced cell invasion; (5) increased activation of caspase-3; (6) improved response to chemotherapy-induced growth inhibition; (6) enhanced Caspase-3 activation and apoptosis induction in combination with chemotherapy and X-ray radiation (chemo/radiosensitization).5. miR-34 restoration inhibits cancer stem cellsCancer cells were stained with CD44/CD133 and sorted by FACS. The CD44+/CD133+ double positive cell population are around 1.5%-2.5%. Tumorsphere culture showed that they had strong tumorsphere formation capability, while single positive cells had very limited tumorsphere formation, but the CD44-/CD133- double negative population did not. The data confirmed that cancer stem cells are in the CD44+/CD133+ population. Real-time PCR data demonstrated that the CD44+/CD133+ double positive population have very low miR-34 expression compared with single positive or CD44-/CD133- double negative populations, suggesting that miR-34 is low in cancer stem cells but increased during differentiation. Restoration of miR-34 significantly inhibited the tumorspheres formation and growth, indicating that miR-34 is able to inhibit the self-renewal of CD44+/CD133+ cancer stem cells and suggesting that miR-34 may become a novel and specific therapy targeting cancer stem cells. 6. Restoration of miR-34 inhibits tumor initiation in nude miceNude mice tumor formation assay using tumorspheres showed much faster tumor initiation and growth than that with total cells inoculation. Restoration of miR-34 significantly inhibited tumor initiation and growth in nude mice than control miRNA.Conclusion:1. miR-34 expression reversely were correlated with tumor aggressiveness and drug-resistance, indicating that miR-34 may be involved in tumor progression as a tumor suppressor gene.2. In p53-deficient tumor, miR-34 inhibited cell growth and invasion and induced G1 block and apoptosis, indicating that miR-34 may restore p53 function, at least to certain extent.3. miR-34 promoted tumor cells growth inhibition and apoptosis induced by chemo/radiotherapy, sensitized tumor cells to chemotherapy and radiation and overcome drug-resistance, which have significant clinical implications.4. CD44+/CD133+ cancer stem cells have much lower miR-34 as compared with differentiated cells; miR-34 restoration inhibited tumorsphere growth or cancer stem cells self-renewal, and inhibited tumor initiation in vivo in nude mice.5. The mechanism of miR-34 mediated suppression of cancer stem cell self-renewal appears related to the direct modulation of downstream targets, Bcl-2, Notch1 and Notch2, which are involved in cancer stem cells regulation, suggesting that miR34 restoration may provide a novel molecular therapy targeting cancer stem cells.
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