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Duke Researchers Find Role for miR-126/miR-126* in Stopping Breast Cancer Metastasis

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A team led by investigators at Duke University Medical Center last month reported on a previously unknown role for a microRNA in combating breast cancer metastasis, publishing data that suggests both strands of the miR-126 duplex simultaneously target a key cytokine to block the entry of metastasis-promoting stromal cells and inflammatory monocytes into the primary tumor microenvironment.

Both miR-126 and the complementary miR-126* are encoded by intron 7 of the epidermal growth-factor-like domain 7, and miR-126 has previously been shown to correlate with poor overall metastasis-free survival in breast cancer patients and to suppress metastasis by targeting angiogenesis-related genes, according to the study, which appeared in Nature Cell Biology.

As part of an effort to uncover the role of miRNAs in how cancer cells interact with their microenvironment, the researchers screened 242 of the non-coding RNAs using a series of human breast cancer cell lines and identified 17 that exhibited “significant expression changes correlated with the metastatic potential of these cells,” including miR-126.

Consistent with previous reports, an analysis of breast cancer patient clinical samples revealed that miR-126 is down-regulated in metastatic tissues compared with primary tumor specimens, with miR-126* exhibiting “a similar pattern,” according to the paper. “Although the miR-126/miR-126* detected in the clinical samples could be of tumor and/or stromal cell origin, together the data indicate that these two microRNAs are potential suppressors of the metastatic process.”

The team then looked to further its findings in vivo, using a retroviral delivery system to generate metastatic breast cancer cells that harbor a vector control or ectopically produce pri-miR-126, which gives rise to both miR-126 and miR-126*. The cells were implanted into a mouse model, and surgically removed 10 days later.

“Consistent with their comparable in vitro proliferation rates, these two cell populations formed primary tumors with a similar size and weight,” the researcher wrote. While ectopic expression of pri-miR-126 had little effect on tumor cell proliferation in vivo, it “significantly suppressed” the formation of lung metastases after the primary tumors were removed.

Using miRNA sponges, the investigators confirmed that suppressing both miRNAs in breast cancer cells significantly boosted their ability to metastasize to the lung.

Notably, when the cancer cells were inoculated by tail vein injection to specifically assess their ability to colonize the lung, the scientists found that over-expression of the miRNAs did not decrease lung metastases.

Thus, in contrast to previous reports, pri-miR-126 may influence the metastatic process through a unique mechanism, “perhaps at the primary tumor site before cancer cells intravasate into the circulation, and through targeting the expression of secreted proteins, such as cytokines and chemokines,” the team suggested.

According to an accompanying review of the findings in Nature Cell Biology, these contradictory results could stem from the differences between cell lines and the mouse models used since miRNAs are “known to function in a context-dependent manner.”

The researchers analyzed cytokine and chemokine expression by cells from the primary tumor and found five whose expression levels were significantly altered. Of these, only one — Sdf-1alpha, also known as Cxcl12 — proved to be a direct target of both miR-126 and miR-126*.

While it is typically only the guide strand of an miRNA duplex that is incorporated into RISC, the investigators discovered that both miR-126 and miR-126* were present in similar numbers in the cancer cells being studied, “suggesting no obvious strand bias between them during RISC loading and protection.”

The team used several control miRNAs to confirm that this deviation from the general rule of miRNA strand selection was not due to the cells themselves, finding that only the designated guiding strand was incorporated into RISC.

“In sharp contrast, miR-126* was incorporated into RISC with a significantly higher efficiency when compared with the star strand of the two control miRNAs,” indicating that both miR-126 and miR-126* are protected by Argonaute proteins and thus able to regulate their target mRNAs, they wrote.

Because Sdf-1alpha mobilizes several cell types, including several that have been implicated in cancer progression, the researchers manipulated the activity of the Sdf-1alpha receptor and miR-126/miR-126* in the cancer cells.

They found that migration of mesenchymal stem cells, which have been reported to promote breast cancer metastasis, was enhanced when the miRNAs were inhibited. Follow-up in vivo experiments showed that ectopic miR-126/miR-126* expression suppressed mesenchymal stem cell recruitment to the primary tumor site.

Meanwhile, the down-regulation of miR-126/miR-126* was also found to increase expression of the chemokine Ccl2, which is known to recruit inflammatory monocytes, in cancer cells. At the same time, Sdf-1alpha suppression blocked the miRNA inhibitory effect, “indicating that suppression of Ccl2 production occurs through an Sdf-1alpha-dependent process,” according to the paper.

According to the review in Nature Cell Biology, the findings collectively suggest a model in which “epigenetic silencing of miR-126/126* promotes breast-to-lung cancer metastasis by the direct de-repression of Sdf-1alpha expression in tumor cells, leading to [mesenchymal stem cell] recruitment to the primary tumor and the subsequent up-regulation of Ccl2 in the tumor cells to attract monocytes to the same site.

“Given the pleiotropic effect of miR-126/126* in inhibiting multiple aspects of tumor growth, angiogenesis, vascular integrity, and metastasis, the prospect of utilizing these miRNAs for cancer therapy is exciting,” the reviewers wrote.

Still, additional studies are needed to evaluate potential adverse effects of miR-126/miR-126* treatment, given that these miRNAs are over-expressed in leukemias and may be involved in the differentiation of megakaryocytes and other leukocytes, they added.

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