Pipeline | miR-34
miR-34 is one of the best characterized tumor suppressor miRNAs to date. It is lost or expressed at reduced levels in numerous cancer types, and the re-introduction of miR-34 mimics inhibits cancer cell growth in vitro and in vivo. miR-34 functions downstream of p53 by regulating genes to induce cell cycle arrest, cellular senescence and apoptosis. Given that >50% of all human cancers show defects in the p53 pathway, miR-34 replacement therapy is likely to become a powerful therapeutic approach (Bader et al., 2010). The following paragraphs illustrate how Mirna scientists identified miR-34 as a critical tumor suppressor in human lung and prostate cancer and how the therapeutic activity was validated in animal models of cancer.
Reduced expression of miR-34 in human tumor tissues
To analyze the expression levels of miR-34 in human cancer tissues, Mirna scientists performed quantitative reverse-transcriptase PCR (qRT-PCR) experiments on RNA samples derived from human lung and prostate tumors (T) as well as their corresponding normal adjacent tissues (NAT). The data reveal that miR-34 was downregulated in the majority of tumor samples: 76% of non-small cell lung cancers and 86% of prostate tumors showed reduced miR-34 expression levels compared to normal lung and prostate (Figure 1; Wiggins et al., 2010). The average miR-34 expression in those samples that showed reduced miR-34 levels was 65% and 57% in lung and prostate tumors, respectively. The data are in agreement with other reports demonstrating reduced miR-34 levels in various cancer types including cancers of the lung, prostate, breast, colon, kidney, bladder, pancreas, ovary, blood and skin (Bommer et al. 2007; Corney et al. 2007; Tazawa et al. 2007; Lodygin et al. 2008; Gallardo et al. 2009; Chim et al. 2010).
Figure 1: Reduced miR-34 expression in prostate and non-small cell lung tumor tissues.
miR-34 expression was determined by quantitative reverse transcriptase PCR using human tumor samples (T) and normal adjacent tissue (NAT) from the same patient. Each line represents expression levels of miR-34 in tumor and NAT from a single patient.
miR-34 replacement eradicates cancer cells in culture
The consistent downregulation of miR-34 across multiple cancer types suggests that reduced miR-34 levels is critical for cancer cell viability and that re-introduction of miR-34 mimics will interfere with the oncogenic properties of cancer cells. To address this experimentally, Mirna scientists administered miR-34 mimics into cultured lung and prostate cancer cells. As shown in Figure 2, repeated exposure of lung and prostate cancer cells to miR-34 mimics strongly inhibited cancer cell growth (see also Wiggins et al., 2010). Unlike cells treated with a “negative control miRNA” (miR-NC), cells treated with miR-34 remained sub-confluent and showed signs of senescence and apoptosis. Many genes regulated by miR-34 encode proteins that function in cell cycle, senescence and apoptosis pathways. Thus, the ability of miR-34 to inhibit cancer cell growth is likely due to a collective inhibition of these target genes.
Figure 2: miR-34 inhibits growth of human lung and prostate cancer cells.
(Left), miR-34 mimics and negative control miRNA (miR-NC) were repeatedly administered to cultured cancer cells on days as indicated by arrows. Cumulative cell numbers are shown in the graphs. (Right), micrographs of cancer cells treated with miR-34 and miR-NC.
miR-34 inhibits lung tumor growth in mice
To test whether miR-34 mimics are able to inhibit tumor growth in vivo, Mirna treated human tumors grown subcutaneously in mice. The miR-34 mimic was formulated in a proprietary neutral lipid emulsion and administered systemically by intravenous tail vein injections. The data showed that repeated administrations of the miR-34 formulation prevented the growth of tumors in mice (Wiggins et al. 2010). In contrast, mice treated with the negative control miRNA developed tumors similar to those treated with saline. A histopathological examination of tumors revealed that miR-34 treatment led to a specific knock-down of miR-34 target genes, reduced proliferation and increased apoptosis in tumor cells (Wiggins et al., 2010).
To further demonstrate the therapeutic activity of the miR-34 mimic, Mirna scientists collaborated with Dr. Slack’s laboratory at Yale University and repeated the experimental framework of the s.c. tumor model in a transgenic mouse model of lung cancer. These animals express an inducible mutant of the K-RAS oncogene that triggers the formation of tumors in the lungs of mice, closely resembling lung tumor development in human patients (Figure 2A). Once mice had developed lung tumors, miR-34 or miR-NC formulated in the neutral lipid emulsion was given repeatedly by intravenous injections. As shown in Figure 2B&C, mice treated with miR-34 developed significantly fewer and smaller malignant lung lesions compared to controls (Trang et al. 2010). The data agree with those obtained from the s.c. tumor model and suggest that miR-34 is a promising approach to treat lung cancer.
Figure 3: Systemic delivery of miR-34 mimics blocks non-small cell lung cancer in the KRAS-G12D transgenic mouse model.
(A) Experimental outline. (B) Lung histologies showing fewer and smaller malignant lesions in mice treated with miR-34 mimics. (C) Quantification of tumor/ total lung area.
miR-34 inhibits metastasis of prostate cancer in mice
In collaboration with Dr. Tang’s laboratory at the University of Texas MD Anderson Cancer Center, Mirna scientists evaluated the therapeutic potential of miR-34 mimics in an orthotopic mouse model of prostate cancer. These tumors produce metastases in various organs leading to death of these animals. Thus, this model can be used to demonstrate enhanced survival provided by the therapeutic. Mice with surgically implanted human prostate cancer xenografts were treated systemically with formulated miR-34. As shown in Figure 5A, miR-34 treatment prolonged survival – at the day of sacrifice, all of the controls and none of the miR-34 treated mice had died (Liu et al., 2011, Nature Medicine). A likely explanation for the increased survival is the observation that miR-34-treated mice displayed ~75% fewer metastatic foci in lung compared to those treated with negative control miRNA (Figure 5B; Liu et al., 2011). Therefore, miR-34 appears to be a promising anti-cancer agent by blocking tumor cell growth and metastasis.
Figure 4: Therapeutic delivery of miR-34 prolongs survival and inhibits metastasis of orthotopic prostate tumors.
(A) Kaplan-Meier curve showing mice carrying orthotopic human prostate cancer xenografts. Four weeks after tumor implantation, mice were treated every other day with formulated miR-34 or controls. (B) Upon sacrifice, lungs from mice treated with either miR-34 or miR-NC were searched for metastatic foci. Total counts are shown.
miR-34 in cancer stem cells
Cancer stem cells are defined as a small fraction of cancer cells that have the ability to self-renew and to give rise to identical daughter cells. They are identified by specific cell surface markers such as CD44 and CD133. Cancer stem cells are often considered to be the “seed” of the tumor and appear to be more tumorigenic, metastatic and refractory to therapy (Reya et al. 2001). Therefore, chemoresistance and recurrence are often explained by the presence of cancer stem cells.
Our knowledge about miRNAs as master regulators of the genome suggests that miRNAs are functional determinants of cellular identity and fate. miRNAs can alter the direction of cellular programs and have been implicated in the “stemness” of normal cells. It is therefore possible that aberrant miRNA function plays also a role in cancer stem cells. To test this hypothesis, scientists from the Tang and Mirna labs determined the expression profile of miRNAs in prostate cancer stem cells and compared it to the bulk of tumor cells. Several miRNAs were found to be differentially expressed, and miR-34 was among the ones that was most significantly downregulated in prostate cancer stem cells (Liu et al., 2011).
To explore whether miR-34 replacement can interfere with the viability of cancer stem cells, the Tang lab performed sphere formation and tumorigenicity assays, both of which indicate phenotypes specifically induced by cancer stem cells. Indeed, transfection of miR-34 mimics inhibited sphere formation by ~50% in several cancer cell lines (Liu et al., 2011). Similarly, the administration of miR-34 via a lentiviral vector into CD44-purified cancer stem cells abolished tumor take in the animal (Figure 5; Liu et al., 2011). The data suggest that reduced miR-34 levels is critical for the viability of prostate cancer stem cells and is in agreement with another report demonstrating a role for miR-34 in cancer stem cells (Ji et al. 2009). Thus, it is likely that therapeutic delivery of miR-34 mimics will not only reduce the bulk of the tumor but also the number of viable cancer stem cells.
Figure 5: miR-34 blocks tumorigenicity of prostate cancer stem cells.
(A) Experimental outline: CD44+ prostate cancer stem cells were purified, infected with lentivirus encoding miR-34 or miR-NC and orthotopically implanted into the dorsal prostate of male mice (10 mice each). (B) Prostate tumors upon sacrifice. In contrast to mice inoculated with miR-NC infected cancer stem cells, none of the miR-34 mice developed tumors.
Summary:
Taken together, Mirna’s drug discovery efforts led to the identification of miR-34 as one of the most central tumor suppressor miRNAs known to date. The frequent downregulation in tumor tissues and the robust inhibitory activity in cancer cells qualify miR-34 as a bona fide tumor suppressor that compares to other well-known protein-encoding tumor suppressor such as p53, PTEN and p16INK4a. In addition, miR-34’s role in cancer stem cells earmarks miR-34 as an ideal therapeutic candidate to combat metastasis, chemoresistance and tumor recurrence. Currently, Mirna is developing several miR-34 mimics toward the clinic to treat patients with solid tumors.
Further reading:
Mirna publications:
The promise of microRNA replacement therapy.
Bader, A. G., D. Brown and M. Winkler (2010). Cancer Res 70(18): 7027-30.
The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.
Liu, C., K. Kelnar, B. Liu, X. Chen, T. Calhoun-Davis, H. Li, L. Patrawala, H. Yan, C. Jeter, S. Honorio, J. F. Wiggins, A. G. Bader, R. Fagin, D. Brown and D. G. Tang (2011). Nat Med 17(2): 211-5.
Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice.
Trang, P., J. F. Wiggins, C. L. Daige, C. Cho, M. Omotola, D. Brown, J. B. Weidhaas, A. G. Bader and F. J. Slack (2010). Mol Ther. EPub ahead of print.
Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.
Wiggins, J. F., L. Ruffino, K. Kelnar, M. Omotola, L. Patrawala, D. Brown and A. G. Bader (2010). Cancer Res 70(14): 5923-30.
Other publications:
p53-mediated activation of miRNA34 candidate tumor-suppressor genes.
Bommer, G. T., I. Gerin, Y. Feng, A. J. Kaczorowski, R. Kuick, R. E. Love, Y. Zhai, T. J. Giordano, Z. S. Qin, B. B. Moore, O. A. MacDougald, K. R. Cho and E. R. Fearon (2007). Curr Biol 17(15): 1298-307.
Epigenetic inactivation of the miR-34a in hematological malignancies.
Chim, C., K. Wong, Y. Qi, F. Loong, W. Lam, L. Wong, D. Jin, J. Costello and R. Liang (2010). Carcinogenesis 31(4):745-50.
Frequent Downregulation of miR-34 Family in Human Ovarian Cancers.
Corney, D. C., C. I. Hwang, A. Matoso, M. Vogt, A. Flesken-Nikitin, A. K. Godwin, A. A. Kamat, A. K. Sood, L. H. Ellenson, H. Hermeking and A. Y. Nikitin (2007). Clin Cancer Res 16(4): 1119-1128.
miR-34a as a prognostic marker of relapse in surgically resected non-small-cell lung cancer.
Gallardo, E., A. Navarro, N. Vinolas, R. M. Marrades, T. Diaz, B. Gel, A. Quera, E. Bandres, J. Garcia-Foncillas, J. Ramirez and M. Monzo (2009). Carcinogenesis 30(11): 1903-9.
MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells.
Ji, Q., X. Hao, M. Zhang, W. Tang, M. Yang, L. Li, D. Xiang, J. T. Desano, G. T. Bommer, D. Fan, E. R. Fearon, T. S. Lawrence and L. Xu (2009). PLoS One 4(8): e6816.
Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.
Lodygin, D., V. Tarasov, A. Epanchintsev, C. Berking, T. Knyazeva, H. Korner, P. Knyazev, J. Diebold and H. Hermeking (2008). Cell Cycle 7(16): 2591-600.
Stem cells, cancer, and cancer stem cells.
Reya, T., S. J. Morrison, M. F. Clarke and I. L. Weissman (2001). Nature 414(6859): 105-11.
Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.
Tazawa, H., N. Tsuchiya, M. Izumiya and H. Nakagama (2007). Proc Natl Acad Sci U S A 104(39): 15472-7.