Tumor suppressor microRNAs (miRNA) provide a new opportunity to treat cancer. This approach, "miRNA replacement therapy," is based on the concept that the reintroduction of miRNAs depleted in cancer ...cells reactivates cellular pathways that drive a therapeutic response. Here, we describe the development of a therapeutic formulation using chemically synthesized miR-34a and a lipid-based delivery vehicle that blocks tumor growth in mouse models of non-small-cell lung cancer. This formulation is effective when administered locally or systemically. The antioncogenic effects are accompanied by an accumulation of miR-34a in the tumor tissue and downregulation of direct miR-34a targets. Intravenous delivery of formulated miR-34a does not induce an elevation of cytokines or liver and kidney enzymes in serum, suggesting that the formulation is well tolerated and does not induce an immune response. The data provide proof of concept for the systemic delivery of a synthetic tumor suppressor mimic, obviating obstacles associated with viral-based miRNA delivery and facilitating a rapid route for miRNA replacement therapy into the clinic.
Cancer stem cells (CSCs), or tumor-initiating cells, are involved in tumor progression and metastasis. MicroRNAs (miRNAs) regulate both normal stem cells and CSCs, and dysregulation of miRNAs has ...been implicated in tumorigenesis. CSCs in many tumors-including cancers of the breast, pancreas, head and neck, colon, small intestine, liver, stomach, bladder and ovary-have been identified using the adhesion molecule CD44, either individually or in combination with other marker(s). Prostate CSCs with enhanced clonogenic and tumor-initiating and metastatic capacities are enriched in the CD44+ cell population, but whether miRNAs regulate CD44+ prostate cancer cells and prostate cancer metastasis remains unclear. Here we show, through expression analysis, that miR-34a, a p53 target, was underexpressed in CD44+ prostate cancer cells purified from xenograft and primary tumors. Enforced expression of miR-34a in bulk or purified CD44+ prostate cancer cells inhibited clonogenic expansion, tumor regeneration, and metastasis. In contrast, expression of miR-34a antagomirs in CD44− prostate cancer cells promoted tumor development and metastasis. Systemically delivered miR-34a inhibited prostate cancer metastasis and extended survival of tumor-bearing mice. We identified and validated CD44 as a direct and functional target of miR-34a and found that CD44 knockdown phenocopied miR-34a overexpression in inhibiting prostate cancer regeneration and metastasis. Our study shows that miR-34a is a key negative regulator of CD44+ prostate cancer cells and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against prostate CSCs.
Abstract
Prostate cancer cells are heterogeneous in their tumorigenicity. For example, the side population cells isolated from LAPC9 xenografts are 100 to 1,000 times more tumorigenic than the ...corresponding non–side population cells. Highly purified CD44+ prostate cancer cells from several xenografts are also enriched in prostate cancer stem/progenitor cells. Because the CD44+ prostate cancer cell population is still heterogeneous, we wonder whether we could further enrich for tumorigenic prostate cancer cells in this population using other markers. Integrin α2β1 has been proposed to mark a population of normal human prostate stem cells. Therefore, we first asked whether the α2β1+/hi cells in prostate tumors might also represent prostate cancer stem cells. Highly purified (≥98%) α2β1+/hi cells from three human xenograft tumors, Du145, LAPC4, and LAPC9, show higher clonal and clonogenic potential than the α2β1−/lo cells in vitro. However, when injected into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse prostate or s.c., the α2β1+/hi prostate cancer cells are no more tumorigenic than the α2β1−/lo cells. Immunofluorescence studies reveal that CD44 and α2β1 identify an overlapping and inclusive population of prostate cancer cells in that ∼70% of α2β1+/hi cells are CD44+ and 20% to 30% of CD44+ cells are distributed in the α2β1−/lo cell population. Subsequently, we sorted out CD44+α2β1+/hi, CD44+α2β1−/lo, CD44−α2β1+/hi, and CD44−α2β1−/lo cells from LAPC9 tumors and carried out tumorigenicity experiments. The results revealed a hierarchy in tumorigenic potential in the order of CD44+α2β1+/hi ≈ CD44+α2β1−/lo > CD44−α2β1+/hi ≫ CD44−α2β1−/lo. These observations together suggest that prostate cancer cells are organized as a hierarchy. Cancer Res 2007;67(14):6796–805
Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer ...cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.
Recently, several human cancers including leukemia and breast and brain tumors were found to contain stem-like cancer cells called cancer stem cells (CSC). Most of these CSCs were identified using ...markers that identify putative normal stem cells. In some cases, stem-like cancer cells were identified using the flow cytometry-based side population technique. In this study, we first show that approximately 30% of cultured human cancer cells and xenograft tumors examined ( approximately 30 in total) possess a detectable side population. Purified side population cells from two cell lines (U373 glioma and MCF7 breast cancer) and a xenograft prostate tumor (LAPC-9) are more tumorigenic than the corresponding non-side population cells. These side population cells also possess some intrinsic stem cell properties as they generate non-side population cells in vivo, can be further transplanted, and preferentially express some "stemness" genes, including Notch-1 and beta-catenin. Because the side population phenotype is mainly mediated by ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, we subsequently studied ABCG2+ and ABCG2- cancer cells with respect to their tumorigenicity in vivo. Although side population cells show increased ABCG2 mRNA expression relative to the non-side population cells and all cancer cells and xenograft tumors examined express ABCG2 in a small fraction (0.5-3%) of the cells, highly purified ABCG2+ cancer cells, surprisingly, have very similar tumorigenicity to the ABCG2- cancer cells. Mechanistic studies indicate that ABCG2 expression is associated with proliferation and ABCG2+ cancer cells can generate ABCG2- cells. However, ABCG2- cancer cells can also generate ABCG2+ cells. Furthermore, the ABCG2- cancer cells form more and larger clones in the long-term clonal analyses and the ABCG2- population preferentially expresses several "stemness" genes. Taken together, our results suggest that (a) the side population is enriched with tumorigenic stem-like cancer cells, (b) ABCG2 expression identifies mainly fast-cycling tumor progenitors, and (c) the ABCG2- population contains primitive stem-like cancer cells.
MicroRNAs have been increasingly implicated in human cancer and interest has grown about the potential to use microRNAs to combat cancer. Lung cancer is the most prevalent form of cancer worldwide ...and lacks effective therapies. Here we have used both in vitro and in vivo approaches to show that the let-7 microRNA directly represses cancer growth in the lung. We find that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using an established orthotopic mouse lung cancer model, we show that intranasal let-7 administration reduces tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings provide direct evidence that let-7 acts as a tumor suppressor gene in the lung and indicate that this miRNA may be useful as a novel therapeutic agent in lung cancer.
Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, ...which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, α2β1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.
Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell (ESC) self‐renewal gene NANOG is purportedly expressed by some epithelial cancer cells but a causal ...role in tumor development has remained unclear. Here, we provide compelling evidence that cultured cancer cells, as well as xenograft‐ and human primary prostate cancer cells express a functional variant of NANOG. NANOG mRNA in cancer cells is derived predominantly from a retrogene locus termed NANOGP8. NANOG protein is detectable in the nucleus of cancer cells and is expressed higher in patient prostate tumors than matched benign tissues. NANOGP8 mRNA and/or NANOG protein levels are enriched in putative cancer stem/progenitor cell populations. Importantly, extensive loss‐of‐function analysis reveals that RNA interference‐mediated NANOG knockdown inhibits tumor development, establishing a functional significance for NANOG expression in cancer cells. Nanog short hairpin RNA transduced cancer cells exhibit decreased long‐term clonal and clonogenic growth, reduced proliferation and, in some cases, altered differentiation. Thus, our results demonstrate that NANOG, a cell‐fate regulatory molecule known to be important for ESC self‐renewal, also plays a novel role in tumor development. Stem Cells 2009;27:993–1005
The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and ...progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays need be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, we present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples.
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