A research team from Beth Israel Deaconess Medical Center this month published two papers implicating a single microRNA, miR-22, in both breast and hematologic cancers, and suggested that its suppression may be an effective therapeutic strategy.
"This is the first time that a microRNA has been shown to drive both cancer initiation and metastasis in a mouse model," Pier Paolo Pandolfi, senior author of the papers and scientific director of the cancer center at Beth Israel, said in a statement. "It's amazing that, by itself, this … microRNA can trigger cancer in two different organs, perhaps in many more, and in the case of breast cancer, can also promote metastasis."
In the first paper, the investigators examined the oncogenic effect of the miRNA by transducing a retroviral vector encoding the human miR-22 gene into immortalized human breast epithelial cells.
They observed an increase in cell motility in vitro, as well as a “dramatic morphological change” in cells overexpressing miR-22 and an increase in the cells’ ability to develop into the mammosphere structures that are indicative of stemness. Taken together, these data suggested that the miRNA triggers the epithelial-mesenchymal transition, or EMT, that enables cancer cells to become migratory and invasive.
They then tried to replicate their findings in vivo by introducing miR-22-expressing retroviral vectors into the mammary tissue of a xenograft mouse model based on a non-metastatic breast cancer cell line. They found that tumors containing the miRNA were highly invasive and metastatic compared with control tumors — a key finding given the non-metastatic nature of the cancer cells tested.
The scientists next moved to confirm their findings using a conditional transgenic mouse model in which miR-22 expression could be induced specifically in mammary glands. In line with the cell line experiments, the team found a “marked increase in EMT features” in cells derived from the animals at a young age.
“Strikingly, by seven weeks of age, miR-22 transgenic mice demonstrated an increased ductal side-branching and higher numbers of mammary stem cells, and cells from these mice displayed an increased ability to form mammospheres,” they wrote in their paper, which appeared in Cell.
When they examined the effect of miR-22 expression in transgenic mice known to develop breast tumors that spontaneously metastasize to the lung, the miRNA proved to increase the incidence and penetrance of metastatic cancer.
Through further experimentation, the researchers demonstrated that miR-22 enhances metastasis by inhibiting the anti-metastatic miRNA miR-200. Specifically, miR-22 directly targets the TET family of methylcytocine dioxygenases, which contribute to DNA demethylation and have been linked to epigenetic reprogramming and cellular differentiation, thereby blocking demethylation of the miR-200 promoter.
In a bid to extend their observations into the clinic, the investigators tested tumor tissue from human breast cancer patients and found a high expression rate of miR-22 in non-triple-negative breast cancer subtypes of breast cancer compared with normal breast tissue.
They also discovered that patients with elevated levels of miR-22 had high-grade tumors of an advanced stage and poor survival rates versus patients with low levels of the miRNA.
In their second paper, which appeared in Cell Stem Cell, the Beth Israel group focused on myelodysplastic syndrome, or MDS, a stem cell disorder that frequently progresses to overt leukemia, finding that miR-22 is “markedly” upregulated in MDS patients and that its aberrant expression “directly correlates with poor survival rates.”
To explore this finding in vivo, the researchers generated a conditional transgenic mouse model that overexpresses the miRNA in the hematopoietic compartment.
The animals showed reduced levels of global 5-hydroxymethylcytosine, the function of which is unknown but which appears to be involved in gene regulation, and increased hematopoietic stem cell self-renewal accompanied by defective differentiation, according to the paper. Over time, they developed MDS and hematological malignancies.
Additionally, blocking miR-22 expression prevented proliferation in both human and mouse leukemic cells.
The team identified TET family member TET2, which is mutated in a variety of blood cancers, as a target of miR-22, and found that overexpression of the miRNA decreased the expression of a number of putative TET2 targets in the hematopoietic stem cell compartment of the transgenic mice. Meanwhile, ectopic expression of TET2 suppressed miR-22-induced phenotypes both in vitro and in vivo.
Turning to MDS patients, the researchers found that around 60 percent displayed a reduction in TET2 levels, which was directly correlated with poor survival rates. About 28 percent of those patients showed elevated miR-22 levels.
In both papers, the Beth Israel investigators tested the use of miR-22 “sponges” with an eye toward blocking the miRNA’s expression for therapeutic purposes.
In the study described in Cell, the sponges were constructed by inserting miR-22-binding sites, arrayed in tandem, into the 3’ UTR of a reporter gene encoding destabilized green fluorescent protein driven by the CMV promoter. The binding sites were perfectly complementary in the seed region, and contained bulges at positions 9 through 12 to prevent degradation.
The sponges led to a reduction in metastatic phenotypes such as invasion abilities in breast cancer cells in vitro, while increasing levels of miR-200 family members. They also “drastically inhibited” in vivo breast cancer metastases to the lung in xenograft models.
In the Cell Stem Cell paper, the team tested both miR-22 retroviral sponges, as well as locked nucleic acids designed to inhibit the miRNA. In human leukemic cells, miR-22 suppression with the LNAs resulted in a “significant reduction in cell proliferation and an elevation of the expression of TET2 and its targets.”
The sponge, meanwhile, led to a reduction in the colony-forming capability of hematopoietic stem/progenitor cells purified from miR-22 transgenic mice.
Overall, the researchers concluded that their data provide a rationale for the therapeutic targeting of miR-22 to prevent EMT and metastatic phenotypes in cancer, and for reactivating TET2 in conditions such as MDS and leukemia.
Given that LNAs are already in the clinic, and that one — Santaris Pharma’s phase II hepatitis C treatment miravirsen — targets a miRNA, the investigators see particular potential for that technology’s use in therapeutically inhibiting miR-22.