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Evaluation of metastatic and angiogenic potential of cancer stem-like cells in distinct mouse models for lung and liver metastasis

Final Report Summary - METASTASIS (Evaluation of metastatic and angiogenic potential of cancer stem-like cells in distinct mouse models for lung and liver metastasis.)

Lung cancer is undoubtedly the most lethal type of cancer worldwide and the 5-year overall survival merely reach 15%, which then drops to 2% when metastasis occur. In the case of colon cancer, it is the fourth leading cause of cancer mortality where the major complications involve the progression through liver metastases. The cancer stem cell (CSC) theory defines a subset of cancer cells with the exclusive ability to drive the growth of a tumor. However, whether the metastatic-initiating cells originate from a CSC population remains unclear. Understanding the mechanisms leading to metastasis initiation and progression is essential for the development of new therapeutic strategies.

General objectives:The aims of this study were to determine the relative contribution of CSCs to metastasis and elucidate the mechanisms involved.

Methodology: To address the first question, we used a mouse model of bone metastasis induced by the intracardiac inoculation of mouse and human lung cancer cells. In the filed, tumor sphere cultures have been widely used to study CSC function. These spherical structures are enriched with cells displaying stem-like properties, namely the overexpression of stem cell markers and the capacity to grow in anchorage independent conditions. We prepared sphere cultures from lung cancer cells and evaluated their metastatic potential as compared to matched monolayer cultures. Representative sphere cultures are shown in Figure 1.

To answer the second objective, we employed a mouse model of colorectal cancer liver metastases. By means of serial transplantation, we obtained liver metastatic variants that displayed extremely strong colonization ability. To determine the gene signature profile of these cells, we performed gene expression array as well as microRNA profiling.


-Bone metastasis
In the first study, we found that spheres overexpressed a variety of stem cell markers, possessed reduced growth kinetic and displayed resistance to conventional therapy. They also presented greater sensitivity to the CSC-targeting drug salinomycin. The CSC phenotype of spheres was confirmed by higher growth ability in soft-agar and tumorigenic potential after sub-cutaneous (s.c.) injection. Surprisingly, while producing large s.c. tumors, the appearance of spontaneous lung metastases was strongly delayed in mice injected with spheres as compared to adherent cells. Such observation was extended by intracardiac inoculation, where spheres showed a dramatic delay in the appearance of metastatic lesions. Assessment of osseous colonization activity demonstrated a strong decrease in bone tumor burden for spheres-injected mice as compared to adherent cells. Interestingly, such delay in colonization was associated with the quiescent phenotype of spheres that displayed a marked cell cycle arrest in the G0/G1 phase, resulting in greater dye-retention ability. Underlined mechanisms included an increase in p27 protein and lower pErk levels. In vitro quiescence was further evidenced in vivo, where both cells types displayed comparable bone homing ability, but with slower growth ability in the case of spheres-injected mice.

-Liver metastasis
In the second study, the comparison of highly metastatic variants with the MC38 parental cell line identified various significantly deregulated genes and microRNAs. microRNAs consist of small non-coding RNAs that regulate the expression of protein-coding genes by binding complementary sequences. They have been shown to participate in carcinogenesis by modulating the expression of tumor-specific oncogenes or tumor suppressors. New studies suggest that microRNAs could also participate in metastatic processes. Here, a predictive analysis between the two arrays highlighted a possible mechanism of control of met proto-oncogene (c-met) by miR-146a. We confirmed the reduced expression of miR-146a in liver metastasis and the elevation of c-met at the mRNA and protein levels. Our results demonstrated that the metastatic-implicated miR-146a directly binds to c-met, impeding its translation.

Of relevance, overexpression of this miR in metastatic cells showed reduced in vitro malignancy. We next examined how restoration of miR-146a in metastatic cells could affects tumor growth and liver metastasis development in mice. Interestingly, while injection of controls cells led to time-dependent increase in total luciferase signal, the level was below the detection limit in miR-146a-expressing cells. When looking at resected organs, mice injected with the metastatic variant displayed large tumor mass in the spleen and the liver, while those from miR-146a-expressing cells clearly presented normal aspect. These data suggest that this microRNA totally abolish primary tumor growth and subsequent liver metastasis.

-Conclusion: Taken together, these data suggest that cells enriched for CSC properties display an increased latency period for the formation of metastatic lesions. Thus, differentiation of CSC in the target organ might be required for the emergence of cell variants with high prometastatic activity. We postulate that microenvironmental factors imposed by each target metastatic organ might be crucial in this process.

In the second part, our results document a new epigenetic mechanism for c-met regulation in colorectal cancer liver metastases and highlight the crucial role of miR-146a in tumorigenesis.

-Potential impact
Metastasis disease is the leading cause of cancer-related mortality with very few effective treatment options available for patients. The spread of cancer cells to remote organs is frequent and can occur at early stages of tumorigenesis. Curiously, a latency period that ranges from years to decades generally takes place between the removal of the primary tumor and the appearance of metastasis. This study offer insights on how such phenomenon might occur and generate resistance to treatment. This could lead to the development on new therapy that could directly target the metastatic-initiating cells and the progression through metastasis outgrow.