Genetic Predictors of Clinical Outcomes in Non-Small Cell Lung Cancer Patients

Lung cancer is the leading cause of cancer-related mortality in the US. Emerging evidence has shown that host genetic factors can interact with environmental exposures to influence patient susceptibility to the diseases as well as clinical outcomes, such as survival and recurrence. We aimed to identify genetic prognostic markers for non-small cell lung cancer (NSCLC), a major (85%) subtype of lung cancer, and also in other subgroups. With the fast evolution of genotyping technology, genetic association studies have went through candidate gene approach, to pathway-based approach, to the genome wide association study (GWAS). Even in the era of GWAS, pathway-based approach has its own advantages on studying cancer clinical outcomes: it is cost-effective, requiring a smaller sample size than GWAS easier to identify a validation population and explore gene-gene interactions. In the current study, we adopted pathway-based approach focusing on two critical pathways - miRNA and inflammation pathways. MicroRNAs (miRNA) post-transcriptionally regulate around 30% of human genes. Polymorphisms within miRNA processing pathways and binding sites may influence patients’ prognosis through altered gene regulation. Inflammation plays an important role in cancer initiation and progression, and also has shown to impact patients’ clinical outcomes. We first evaluated 240 single nucleotide polymorphisms (SNPs) in miRNA biogenesis genes and predicted binding sites in NSCLC patients to determine associations with clinical outcomes in early-stage (stage I and II) and late-stage (stage III and IV) lung cancer patients, respectively. First, in 535 early-stage patients, after correcting multiple comparisons, FZD4:rs713065 (hazard ratio [HR]:0.46, 95% confidence interval [CI]:0.32-0.65) showed a significant inverse association with survival in early stage surgery-only patients. SP1:rs17695156 (HR:2.22, 95% CI:1.44-3.41) and DROSHA:rs6886834 (HR:6.38, 95% CI:2.49-16.31) conferred increased risk of progression in the all patients and surgery-only populations, respectively. FAS:rs2234978 was significantly associated with improved survival in all patients (HR:0.59, 95% CI:0.44-0.77) and in the surgery plus chemotherapy populations (HR:0.19, 95% CI:0.07-0.46).. Functional genomics analysis demonstrated that this variant creates a miR-651 binding site resulting in altered miRNA regulation of FAS, providing biological plausibility for the observed association. We then analyzed these associations in 598 late-stage patients. After multiple comparison corrections, no SNPs remained significant in the late stage group, while the top SNP NAT1:rs15561 (HR=1.98, 96%CI=1.32-2.94) conferred a significantly increased risk of death in the chemotherapy subgroup. To test the hypothesis that genetic variants in the inflammation-related pathways may be associated with survival in NSCLC patients, we first conducted a three-stage study. In the discovery phase, we investigated a comprehensive panel of 11,930 inflammation-related SNPs in three independent lung cancer populations. A missense SNP (rs2071554) in HLA-DOB was significantly associated with poor survival in the discovery population (HR: 1.46, 95% CI: 1.02-2.09), internal validation population (HR: 1.51, 95% CI: 1.02-2.25), and external validation (HR: 1.52, 95% CI: 1.01-2.29) population. Rs2900420 in KLRK1 was significantly associated with a reduced risk for death in the discovery (HR: 0.76, 95% CI: 0.60-0.96) and internal validation (HR: 0.77, 95% CI: 0.61-0.99) populations, and the association reached borderline significance in the external validation population (HR: 0.80, 95% CI: 0.63-1.02). We also evaluated these inflammation-related SNPs in NSCLC patients in never smokers. Lung cancer in never smokers has been increasingly recognized as distinct disease from that in ever-smokers. A two-stage study was performed using a discovery population from MD Anderson (411 patients) and a validation population from Mayo Clinic (311 patients). Three SNPs (IL17RA:rs879576, BMP8A:rs698141, and STK:rs290229) that were significantly associated with survival were validated (pCD74:rs1056400 and CD38:rs10805347) were borderline significant (p=0.08) in the Mayo Clinic population. In the combined analysis, IL17RA:rs879576 resulted in a 40% reduction in the risk for death (p=4.1 × 10-5 [p=0.61, heterogeneity test]). We also validated a survival tree created in MD Anderson population in the Mayo Clinic population. In conclusion, our results provided strong evidence that genetic variations in specific pathways that examined (miRNA and inflammation pathways) influenced clinical outcomes in NSCLC patients, and with further functional studies, the novel loci have potential to be translated into clinical use.

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.

E2F1 AND TUMOR SUPPRESSION: THE ROLE OF p21, miRNAS, AND THE DNA DAMAGE RESPONSE

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation through a non-apoptotic mechanism requiring ARF and p53, but not p21. However, p21-loss inhibited two-stage chemical carcinogenesis in FVB mice. In response to E2F1 overexpression, we found that 22 miRNAs are differentially regulated in mouse epidermis, including let-7a, let-7c, and miR-301. Additionally, regulation of miR-301 involves binding of E2F1 to its promoter. Finally, our data indicate a role for E2F1 at sites of DNA damage requiring E2F1’s phosphorylation at serine 31 which may involve DNA repair. Further, this role in the DDR may affect tumor aggressiveness and multiplicity. In all, we have explored three mechanisms for E2F1-induced tumor suppression and identified E2F1’s role in the DNA damage response as a likely contributor to this phenomenon.

ΔNp63 REGULATES A COMPLEX NETWORK OF TARGET GENES IN LIMB AND EPIDERMAL DEVELOPMENT

The skin is composed of two major compartments, the dermis and epidermis. The epidermis forms a barrier to protect the body. The stratified epithelium has self-renewing capacity throughout life, and continuous turnover is mediated by stem cells in the basal layer. p63 is structurally and functionally related to p53. In spite of their structural similarities, p63 is critical for the development and maintenance of stratified epithelial tissues, unlike p53. p63 is highly expressed in the epidermis and previously has been shown to play a critical role in the development and maintenance of the epidermis. The study of p63 has been complicated due to the existence of multiple isoforms: those with a transactivation domain (TAp63) and those lacking this domain (ΔNp63). Mice lacking p63 cannot form skin, have craniofacial and skeletal defects and die within hours after birth. These defects are due to the ability of p63 to regulate multiple processes in skin development including epithelial stem cell proliferation, differentiation, and adherence programs. To determine the roles of these isoforms in skin development and maintenance, isoform specific p63 conditional knock out mice were generated by our lab. TAp63-/- mice age prematurely, develop blisters, and display wound-healing defects that result from hyperproliferation of dermal stem cells. That results in premature depletion of these cells, which are necessary for wound repair, that indicates TAp63 plays a role in dermal/epidermal maintenance. To study the role of ΔNp63, I generated a ΔNp63-/- mouse and analyzed the skin by performing immunofluorescence for markers of epithelial differentiation. The ΔNp63-/- mice developed a thin, disorganized epithelium but differentiation markers were expressed. Interestingly, the epidermis from ΔNp63-/- mice co-expressed K14 and K10 in the same cell suggesting defects in epidermal differentiation and stratification. This phenotype is reminiscent of the DGCR8fl/fl;K14Cre and Dicerfl/fl;K14Cre mice skin. Importantly, DGCR8-/- embryonic stem cells (ESCs) display a hyperproliferation defect by failure to silence pluripotency genes. Furthermore, I have observed that epidermal cells lacking ΔNp63 display a phenotype reminiscent of embryonic stem cells instead of keratinocytes. Thus, I hypothesize that genes involved in maintaining pluripotency, like Oct4, may be upregulated in the absence of ΔNp63. To test this, q-RT PCR was performed for Oct4 mRNA with wild type and ΔNp63-/- 18.5dpc embryo skin. I found that the level of Oct4 was dramatically increased in the absence of ΔNp63-/-. Based on these results, I hypothesized that ΔNp63 induces differentiation by silencing pluripotency regulators, Oct4, Sox2 and Nanog directly through the regulation of DGCR8. I found that DGCR8 restoration resulted in repression of Oct4, Sox2 and Nanog in ΔNp63-/- epidermal cells and rescue differentiation defects. Loss of ΔNp63 resulted in pluripotency that caused defect in proper differentiation and stem cell like phenotype. This led me to culture the ΔNp63-/- epidermal cells in neuronal cell culture media in order to address whether restoration of DGCR8 can transform epidermal cells to neuronal cells. I found that DGCR8 restoration resulted in a change in cell fate. I also found that miR470 and miR145 play a role in the induction of pluripotency by repressing Oct4, Sox2 and Nanog. This indicates that ΔNp63 induces terminal differentiation through the regulation of DGCR8.

Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

The Role of Cancer-Associated Fibroblasts in Lung Tumorigenesis

The extracellular milieu is rich in growth factors that drive tumor progression,but the mechanisms that govern tumor cell sensitivity to those ligands have notbeen fully defined. In this study, we address this question in mice that developmetastatic lung adenocarcinomas through the suppression of the microRNA-200 (miR-200) family. Cancer-associated fibroblasts (CAF) enhance tumorgrowth and invasion by secreting VEGF-A that binds to VEGFR1, a processrequired for tumor growth and metastasis in mice and correlated with a poorprognosis in lung adenocarcinoma patients. In this study, we discovered thatmiR-200 blocked CAF-induced tumor cell invasion by directly targetingVEGFR1 in tumor cells. In the context of previous studies, our findings suggestthat the miR-200 family is a point of convergence for diverse biologic processesthat regulate tumor cell proliferation, invasion, and metastasis; its target genesixdrive epithelial-to-mesenchymal transition (ZEB1 and ZEB2) and promotesensitivity to a potent tumor growth factor emanating from the microenvironment(VEGFR1). Clinical trials should focus not only on the role of VEGFR1 inangiogenesis but also on the expression and activation of VEGFR1 in tumorcells by stromal sources of VEGF-A in the tumor microenvironment as a targetfor metastasis prevention.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

Micro RNA expression alterations in patients with glioblastoma

Glioblastoma, also known as glioblastoma multiform or GBM, is the most common and most malignant primary brain tumor. The clinical history of patients with glioblastoma is short, usually less than 3 months in more than 50% of cases after diagnosis. Currently, the methods of glioblastoma treatment are chemotherapy, radiotherapy and surgery. Even with the more effective treatment options, patients with glioblastoma most likely have a median survival time of 10 to 12 months. It is necessary to seek other treatment methods, including gene-targeted treatment. The success of gene-targeted treatment depends critically on the knowledge of genes that may be the cause of, or contribute to disease. To establish a correlate between glioblastoma survival timeline and micro RNA expression alteration, a study of 91 glioblastoma patients was conducted at the University of Texas M. D. Anderson Cancer Center. These 91 glioblastoma patients were newly diagnosed from 2002 to 2007. Statistical analysis was conducted to test the association of miRNA expression alteration between long-term survival and short-term survival glioblastoma. The completion of this proposed study will provide a better understanding of the regulatory role of miRNA in glioblastoma progression.^

Transcriptional and translational regulators of gene expression in germ cell development

Germ cell development is a highly coordinated process driven, in part, by regulatory mechanisms that control gene expression. Not only transcription, but also translation, is under regulatory control to direct proper germ cell development. In this dissertation, I have focused on two regulators of germ cell development. One is the homeobox protein RHOX10, which has the potential to be both a transcriptional and translational regulator in mouse male germ cell development. The other is the RNA-binding protein, Hermes, which functions as a translational regulator in Xenopus laevis female germ cell development. ^ Rhox10 is a member of reproductive homeobox gene X-(linked (Rhox) gene cluster, of which expression is developmentally regulated in developing mouse testes. To identify the cell types and developmental stages in which Rhox10 might function, I characterized its temporal and spatial expression pattern in mouse embryonic, neonatal, and adult tissues. Among other things, this analysis revealed that both the level and the subcellular localization of RHOX10 are regulated during germ cell development. To understand the role of Rhox10 in germ cell development, I generated transgenic mice expressing an artificial microRNA (miRNA) targeting Rhox10. While this artificial miRNA robustly downregulated RHOX10 protein expression in vitro, it did not significantly reduce RHOX10 expression in vivo. So I next elected to knockdown RHOX10 levels in spermatogonial stem cells (SSCs), which I found highly express both Rhox10 mRNA and RHOX10 protein. Using a recently developed in vitro culture system for SSCs combined with a short-hairpin RNA (shRNA) approach, I strongly depleted RHOX10 expression in SSCs. These RHOX10-depleted cells exhibited a defect in the ability to form stem cell clusters in vitro. Expression profiling analysis revealed many genes regulated by Rhox10, including many meiotic genes, which could be downstream of Rhox10 in a molecular pathway that controls SSC differentiation. ^ RNA recognition motif (RRM) containing protein, Hermes is localized in germ plasm, where dormant mRNAs are also located, of Xenopus oocytes, which implicates its role in translational regulator. To understand the function of Hermes in oocyte meiosis, I used a morpholino oligonucleotide (MO) based knockdown approach. Microinjection of Hermes MO into fully grown oocytes, which are arrested in meiotic prophase, caused acceleration of oocytes reentry into meiosis (i.e., maturation) upon progesterone induction. Using a candidate approach, I identified at least three targets of Hermes: Ringo/Spy, Xcat2, and Mos. Ringo/Spy and Mos are known to have functions in oocyte maturation, while Ringo/Spy, Xcat2 mRNA are localized in the germ plasm of oocytes, which drives germ cell specification after fertilization. This led me to propose that Hermes functions in both oocyte maturation and germ cell development through its ability to regulate 3 crucial target mRNAs. ^

Identification of micro-RNAs targeting genes of PI3K/AKT pathway in ovarian cancer

Ovarian cancer is the leading cause of cancer-related death for females due to lack of specific early detection method. It is of great interest to find molecular-based biomarkers which are sensitive and specific to ovarian cancer for early diagnosis, prognosis and therapeutics. miRNAs have been proposed to be potential biomarkers that could be used in cancer prevention and therapeutics. The current study analyzed the miRNA and mRNA expression data extracted from the Cancer Genome Atlas (TCGA) database. Using simple linear regression and multiple regression models, we found 71 miRNA-mRNA pairs which were negatively associated between 56 miRNAs and 24 genes of PI3K/AKT pathway. Among these miRNA and mRNA target pairs, 9 of them were in agreement with the predictions from the most commonly used target prediction programs including miRGen, miRDB, miRTarbase and miR2Disease. These shared miRNA-mRNA pairs were considered to be the most potential genes that were involved in ovarian cancer. Furthermore, 4 of the 9 target genes encode cell cycle or apoptosis related proteins including Cyclin D1, p21, FOXO1 and Bcl2, suggesting that their regulator miRNAs including miR-16, miR-96 and miR-21 most likely played important roles in promoting tumor growth through dysregulated cell cycle or apoptosis. miR-96 was also found to directly target IRS-1. In addition, the results showed that miR-17 and miR-9 may be involved in ovarian cancer through targeting JAK1. This study might provide evidence for using miRNA or miRNA profile as biomarker.^

MicroRNA Regulation of Prostate Cancer Stem/Progenitor Cells and Prostate Cancer Development

Most human tumors contain a population of cells with stem cell properties, called cancer stem cells (CSCs), which are believed to be responsible for tumor establishment, metastasis, and resistance to clinical therapy. It’s crucial to understand the regulatory mechanisms unique to CSCs, so that we may design CSC-specific therapeutics. Recent discoveries of microRNA (miRNA) have provided a new avenue in understanding the regulatory mechanisms of cancer. However, how miRNAs may regulate CSCs is still poorly understood. Here, we present miRNA expression profiling in six populations of prostate cancer (PCa) stem/progenitor cells that possess distinct tumorigenic properties. Six miRNAs were identified to be commonly and differentially expressed, namely, four miRNAs (miR-34a, let-7b, miR-106a and miR-141) were under-expressed, and two miRNAs (miR-301 and miR-452) were over-expressed in the tumorigenic subsets compared to the corresponding marker-negative subpopulations. Among them, the expression patterns of miR-34, let-7b, miR-141 and miR-301 were further confirmed in the CD44+ human primary prostate cancer (HPCa) samples. We then showed that miR-34a functioned as a critical negative regulator in prostate CSCs and PCa development and metastasis. Over-expression of miR-34a in either bulk or CD44+ PCa cells significantly suppressed clonal expansion, tumor development and metastasis. Systemic delivery of miR-34a in tumor-bearing mice demonstrated a potent therapeutic effect again tumor progression and metastasis, leading to extended animal survival. Of great interest, we identified CD44 itself as a direct and relevant downstream target of miR-34a in mediating its tumor-inhibitory effects. Like miR-34a, let-7 manifests similar tumor suppressive effects in PCa cells. In addition, we observed differential mechanisms between let-7 and miR-34a on cell cycle, with miR-34a mainly inducing G1 cell-cycle arrest followed by cell senescence and let-7 inducing G2/M arrest. MiR-301, on the other hand, exerted a cell type dependent effect in regulating prostate CSC properties and PCa development. In summary, our work reveals that the prostate CSC populations display unique miRNA expression signatures and different miRNAs distinctively and coordinately regulate various aspects of CSC properties. Altogether, our results lay a scientific foundation for developing miRNA-based anti-cancer therapy.

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition