Case study: let-7
The let-7 miRNA plays an important role in lung tumorigenesis making the small RNA one of Mirna’s lead candidates. The following paragraphs show data from Mirna’s let-7 R&D program and will illustrate how restoring normal levels of a miRNA that becomes absent in cancer aids in cancer treatment. This concept of “miRNA Replacement Therapy” may become a powerful approach for future therapeutic regimes against cancer.
We have used miRNA arrays and quantitative real-time polymerase chain reaction (qRT-PCR) assays to compare the expression of miRNAs in tumor (T) and normal adjacent tissues (NAT) from 30 patients with NSCLC, including 9 adenocarcinomas, 17 squamous cell carcinomas and 4 large cell carcinomas. These studies have identified miRNAs whose levels are consistently altered in lung cancer. Among these miRNAs is let-7, which on average exhibits a 54-70% reduction in expression in the tumors of lung cancer patients relative to normal lung tissue from the same patients (Figure 1). The suppression of let-7 was evident in all three major forms of NSCLC: adenocarcinomas, squamous and large cell carcinomas. Our observations, which are in agreement with results from other laboratories, demonstrate that let-7 expression is frequently lost or reduced in NSCLC (Calin et al., 2004; Volinia et al., 2006; Yanaihara et al., 2006). In addition, decreased let-7 levels correlate with poor survival (Yanaihara et al., 2006).
Figure 1: Expression of the let-7 miRNA is reduced in various human NSCLC specimens.
let-7 levels were determined by qRT-PCR in the tumor and the normal adjacent tissue (NAT) from a total of 30 lung cancer patients. Expression of let-7 in the tumor tissue was normalized to the expression in the corresponding NAT (100%). n, number of tumor/NAT sample pairs.
The magnitude and frequency of let-7 down-regulation indicated that let-7 is a causal component of lung cancer development. Therefore, restoring let-7 expression by administering a synthetic let-7 mimetic to lung cancer cells is likely to interfere with the cancerous phenotype. To explore this possibility, we introduced let-7 mimetics into cultured lung cancer cells that originated from 10 different lung cancer patients. Cancer models tested include 7 adenocarcinomas, 2 squamous cell carcinomas and one large cell carcinoma. As shown in Figure 2, temporal or extended treatment induced a robust therapeutic response and decreased the viability of NSCLC cells. The fact that multiple cancer models of NSCLC responded to let-7 treatment suggests that therapeutic applications of let-7 may provide benefits to a broad group of lung cancer patients.
Figure 2: let-7 inhibits proliferation of human lung cancer cells.
(A) A total of 10 NSCLC cell lines, classified by histology into adenocarcinoma, squamous cell carcinoma and large cell carcinoma, were exposed to let-7 mimetic (red) or a “negative control miRNA” (gray, miR-NC) for 3 days. miR-NC chemically resembles an endogenous miRNA, however, is unrelated to any known human genomic sequences and has no effect on cellular properties. Proliferation of negative control-treated cells was set at 100%. (B)) Three independent groups of cultured squamous cell carcinoma cells were treated repeatedly with let-7 or negative control miRNA (miR-NC) on days 0, 7 and 14. Cell counts were obtained on days 7, 14 and 27, averaged and plotted over time. Standard deviations are shown in the graph.
In collaboration with Dr. Frank Slack’s laboratory at Yale University, New Haven, CT., Mirna scientists tested the anti-tumor activity of let-7 in a transgenic mouse model of lung cancer. These animals express an inducible mutant of the K-RAS oncogene that triggers the formation of NSCLC in mice, closely resembling lung tumor development in human patients (Figure 3A). Treatment with a negative control miRNA (scr) resulted in the formation of mouse lung cancer as expected. In contrast, intranasal delivery of let-7 via an adenoviral vector strongly inhibited the formation of lung tumors (Figures 3B-D). The data provide proof-of-concept for the therapeutic utility of let-7 based treatment regimes.
Figure 3: Exogenous let-7 administration reduces lung tumor formation in an orthotopic lung cancer mouse model.
(A) A diagram showing the principle of the conditional mouse model for lung cancer. Intranasal instillation of adenovirus encoding the cre recombinase in transgenic K-RAS G12D mice induces expression of the K-RAS mutant G12D and lung tumors. Concurrent administration of adenovirus encoding let-7 or a negative control miRNA (scr, scrambled) was done to evaluate the anti-tumor effect of let-7. (B,C) Histologies of lungs from K-RAS G12D mice treated with either cre/let-7 or cre/scr are shown. Of note, mice that received cre/let-7 developed much fewer and smaller tumors. Arrowheads indicate areas of alveolar hyperplasia. (D) Quantitative analysis of tumor burden in K-RAS G12D animals treated with cre/let-7 or cre/let-7 (8 animals per group). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. Boxes represent inter-quartile ranges (between the 25th and 75th quartiles) and the two-tailed p-value is indicated. The total range, mean, and median are shown.
Mirna Therapeutics is exploiting the therapeutic potential of let-7 for the development of synthetic let-7 mimetics to combat lung cancer. To test our drug directly in pre-clinical animal studies, we administered let-7 miRNA to human lung cancer xenografts. As shown in Figure 4, the let-7 drug inhibited tumor formation when applied as a single dose to cultured cells immediately prior to injection into animals (panel A). let-7 also blocked tumor growth when it was directly injected into tumors that had developed prior to treatment (panel B). Our pre-clinical data show robust efficacy of the let-7 therapeutic, providing evidence for the importance of miRNAs in the development of cancer and supporting the concept of “miRNA replacement therapy” as an important component of effective cancer treatment regimens of the future.
Figure 4: Local delivery of let-7 inhibits growth of human lung tumors in mice.
(A) Adenocarcinoma cells were treated with let-7 mimetics and implanted into the flank of four immunodeficient mice (ex vivo delivery method). As controls, adenocarcinoma cells were treated with negative control miRNA (miR-NC) and implanted into the opposite flank of these animals. Tumor size measurements were taken periodically over the course of the following 30 days. (B) Large cell carcinoma cells were inoculated subcutaneously into 12 mice and housed until they developed palpable tumors. On days 11, 14 and 17, the let-7 drug was directly injected into tumors of a group of 6 mice carrying large cell carcinomas. A control group of 6 animals received negative control miRNA (miR-NC) following the same dosing schedule. miR-NC treated tumors developed at an equal pace as untreated xenografts (data not shown). Caliper measurements were taken on days as indicated and averaged. Standard deviations are shown in the graphs.
How does let-7 work?
In collaboration with Dr. Frank Slack’s laboratory, Mirna scientists discovered that let-7 directly represses the RAS oncogene. RAS is known to be expressed at elevated levels in NSCLC and is one of the most important oncogenic components that induce lung tumorigenesis. The downregulation of let-7 in lung tumors now provides a clear explanation for increased RAS expression in lung tumors. Our discovery was the first of its nature and proves that miRNAs are directly involved in cancer development by regulating bona fide oncogenes and tumor suppressors. These results were published in 2005 in the journal Cell (Johnson et al., 2005).
Since then, Mirna scientists and others have identified many other genes that are regulated by the let-7 miRNA (Figure 5). Among these are the HMGA2 and Myc oncogenes, as well as cyclin-dependent kinases that contribute to the development of cancer. Since many of these genes function in various pathways that are commonly mis-regulated in cancer, let-7 is able to interfere with multiple cancer-associated pathways, such as mitotic signaling, cell cycle progression and angiogenesis. Considering that current cancer treatment regimes are directed toward single targets – including let-7 targets – the therapeutic benefit of let-7, and miRNAs in general, may be superior.
Figure 5: let-7 regulates multiple cancer-associated pathways
Summary:
Our data demonstrate that administration of let-7 reduces growth of human lung cancer cells in vitro and in pre-clinical animal studies. The enthusiasm for therapeutic development of let-7 is heightened by the fact that it is a naturally occurring molecule with biological implications in cancer that support the concept of “miRNA Replacement Therapy”. Therefore, let-7 therapy might have less unwanted side effects than other chemotherapeutics. Recent observations suggest that let-7 synergizes with conventional chemotherapies and radiation treatments (Ovcharenko et al., 2007; Weidhaas et al., 2007) and also induces a therapeutic response in other cancer types, including prostate cancer and acute myeloid leukemia. Thus, let-7 is an exceptionally promising candidate for further clinical development.
Further reading:
Mirna publications:
Johnson, S. M., H. Grosshans, J. Shingara, M. Byrom, R. Jarvis, A. Cheng, E. Labourier, K. L. Reinert, D. Brown, and F. J. Slack. 2005. RAS is regulated by the let-7 microRNA family. Cell 120:635-47.
Johnson, C. D., A. Esquela-Kerscher, G. Stefani, M. Byrom, K. Kelnar, D. Ovcharenko, M. Wilson, X. Wang, J. Shelton, J. Shingara, L. Chin, D. Brown, and F. J. Slack. 2007. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67:7713-22.
Ovcharenko, D., K. Kelnar, C. Johnson, N. Leng, and D. Brown. 2007. Genome-scale microRNA and small interfering RNA screens identify small RNA modulators of TRAIL-induced apoptosis pathway. Cancer Res. 67:10782-8.
Esquela-Kerscher, A., P. Trang, J. F. Wiggins, L. Patrawala, A. Cheng, L. Ford, J. B. Weidhaas, D. Brown, A. G. Bader, and F. J. Slack. 2008. The let-7 microRNA reduces tumor growth in mouse models of lung cancer.
Cell Cycle 7.
Other publications:
Calin, G. A., C. Sevignani, C. D. Dumitru, T. Hyslop, E. Noch, S. Yendamuri, M. Shimizu, S. Rattan, F. Bullrich, M. Negrini, and C. M. Croce. 2004. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 101:2999-3004.
Volinia, S., G. A. Calin, C. G. Liu, S. Ambs, A. Cimmino, F. Petrocca, R. Visone, M. Iorio, C. Roldo, M. Ferracin, R. L. Prueitt, N. Yanaihara, G. Lanza, A. Scarpa, A. Vecchione, M. Negrini, C. C. Harris, and C. M. Croce. 2006. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 103:2257-61.
Yanaihara, N., N. Caplen, E. Bowman, M. Seike, K. Kumamoto, M. Yi, R. M. Stephens, A. Okamoto, J. Yokota, T. Tanaka, G. A. Calin, C. G. Liu, C. M. Croce, and C. C. Harris. 2006. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 9:189-98.
Mayr, C., M. T. Hemann, and D. P. Bartel. 2007. Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315:1576-9.
Weidhaas, J. B., I. Babar, S. M. Nallur, P. Trang, S. Roush, M. Boehm, E. Gillespie, and F. J. Slack. 2007. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy. Cancer Res.67:11111-6.
Yu, F., H. Yao, P. Zhu, X. Zhang, W. Pan, C. Gong, Y. Huang, S. Hu, F. Su, J. Lieberman, and E. Song. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131:1109-23.
Kumar, M. S., S. J. Erkeland, R. E. Pester, C. Y. Chen, M. S. Ebert, P. A. Sharp, and T. Jacks. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci U S A 105:3903-8.
Review article:
Esquela-Kerscher, A., and F. J. Slack. 2006. Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6:259-69.