Pipeline | let-7
The miRNA let-7 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 illustrate how replacing an endogenous miRNA that has been depleted in cancer is a powerful therapeutic strategy. This concept of “miRNA Replacement Therapy” is likely to become a standard cancer therapy.
Mirna 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 non-small cell lung carcinoma (NSCLC), including 9 adenocarcinomas, 17 squamous cell carcinomas and 4 large cell carcinomas. These studies 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 (see also Johnson et al., 2005. Cell). Our observations were confirmed by results from other laboratories, demonstrating that let-7 expression is frequently lost or reduced in NSCLC (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 indicates that let-7 plays a key role in lung cancer development. Therefore, restoring let-7 expression by administering a synthetic let-7 mimic to lung cancer cells is likely to interfere with the cancerous phenotype. To test this hypothesis, Mirna scientists introduced let-7 mimics into cultured human lung cancer cells. As shown in Figure 2, repeated administration of the let-7 mimic robustly inhibited the proliferation and viability of NSCLC cells and suggests that let-7 functions as a tumor suppressor in lung cancer.
Figure 2: let-7 inhibits proliferation of human lung cancer cells.
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. miR-NC, which chemically resembles an endogenous miRNA, is unrelated to any known human genomic sequences and has no effect on cellular properties.
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 (Esquela-Kerscher et al., 2008).
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 far 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 mimics to combat lung cancer. To test the drug directly in pre-clinical animal studies, Mirna scientists administered let-7 miRNA to human lung cancer xenografts in mice. As shown in Figure 4, the let-7 drug inhibited tumor formation when applied as a single dose immediately prior to
implantation of the tumor xenograft 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; published in Trang et al., 2009). The 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 mimics 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 down-regulation of let-7 in lung tumors now provides a clear explanation for increased RAS expression in lung tumors. This 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 let-7 (Figure 5). Among these are the HMGA2 and Myc oncogenes (Mayr et al., 2007; Sampson et al., 2007), as well as cyclin-dependent kinases that contribute to the development of cancer (Johnson et al., 2007). 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:
Mirna’s data demonstrate that administration of let-7 reduces growth of human lung cancer cells in vitro and in pre-clinical animal studies, and that inadvertent introduction of miRNAs such as let-7 in normal cells does not have toxic effects. Therefore, let-7 therapy will likely have fewer 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.
Trang, P., Medina, P. P., Wiggins, J. F., Ruffino, L., Kelnar, K., Omotola, M., Homer, R., Brown, D., Bader, A. G., Weidhaas, J. B., and F. J. Slack. 2009.
Regression of murine lung tumors by the let-7 microRNA. Oncogene.
Other publications:
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.
Sampson, V. B., Rong, N. H., Han, J., Yang, Q., Aris, V., Soteropoulos, T., Petrelli, N. J., Dunn, S. P., and Krueger, L. J. 2007. MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res. 67:9762-70.
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.