Micro RNAs (miRNAs) are ~20 base pair double-stranded RNA molecules that bind to the 3’ untranslated region of their messenger RNA (mRNA) targets. In so doing, they regulate protein synthesis by causing either translational arrest or RNA degradation. This allows cells to rapidly adapt to changes in their environment, such as the introduction of chemotherapy agents. miRNAs may have important roles in mechanisms for cancer cells to develop resistance to these agents.
Jingfang Ju, Ph.D., studies the relationship between miRNAs and cancer. In a recent webinar, the Stony Brook University Renaissance School of Medicine pathology professor presented findings from his lab’s 15-year study of these small non-coding RNA molecules mediating translational control of genes involved in cell proliferation, cell cycle, autophagy, epithelial-mesenchymal transition (EMT), and cell death. He showed how miRNAs promote, or hinder, natural mechanisms to fight cancer, and how they can interfere or synergize with the ability of anticancer chemotherapies to accomplish their tasks. The webinar culminated with Ju’s exploration of miRNAs as cancer therapies and prognostic biomarkers.
p53
It’s well known that p53 is an important player in many cancers, affecting multiple pathways through multiple mechanisms. The gene for this tumor suppressor is mutated or deleted in more cancers than any other gene, including more than half of all colon cancers. The p53 protein can bind to mRNA, post-transcriptionally affecting a number of genes. It is also a transcription factor, affecting which genes are transcribed in the first place.
Among p53’s many target genes are miRNAs that themselves post-transcriptionally repress target mRNA. In fact, nearly half of putative miRNA promoters contain a potential p53 binding site. miRNAs, many as part of the p53 network, are responsible for keeping cells in check through a wide and varied host of processes. “Through miRNA p53 exerts its cellular function, such as cell cycle regulator and regulating apoptosis,” Ju said.
For example, genotoxic stress, such as exposure to the chemotoxic agents 5-fluorouracil (5-FU) and methotrexate (MTX), upregulates p53, leading to the upregulation of the miRNA miR-192. This, in turn downregulates the expression of target proteins dihydrofolate reductase (DHFR) and thymidylate synthase (TS)—both of which are needed for the cell to produce dTMP, a building block of DNA—“and in so doing regulates cell cycle and cell death.” In this sense, miR-192 and its kin can be considered tumor suppressors. And because DHFR is a target of MTX, and TS is a target of 5-FU, such pathways can enhance the cells’ sensitivity to chemotherapeutics.
Chemoresistance
A tumor suppressor miRNA may not always be a good thing. “The only therapeutic window for chemotherapy is when the tumor cells are rapidly proliferating compared to normal cells,” Ju argues. Most therapeutic compounds are actually very effective at eliminating the rapidly proliferating cells—the trouble is the resistance is coming from slow proliferating stem-like tumor cells. Restoration of cell cycle checkpoints allows the stem-like cells to stop cell cycle and effectively wait out the storm. “This, we believe, is a major contributing factor of chemoresistance mediated by miR-192.”
On the flip side, suppression of some miRNAs is known to be associated with colon cancers. For example, there is a progressive loss of miR-129 expression as tumor tissues are sampled from stage 1 to stage 2 to stage 3 to stage 4. “By stage 4 disease, most of the colon cancer patients have lost the entire miR-129 expression. This was regulated by promoter methylation,” Ju said. “And we know that stage 4 colon cancer is highly resistant to chemo.”
Ju’s lab developed a colon cancer stem-like cell culture by harvesting cells resistant to 5-FU treatment. In subsequent experiments about 80% of the cells were viable even after treatment with 100 µM 5-FU.
miRNA therapy
miR-129 regulates several key targets in colon cancer including the 5-FU target TS, the cell cycle regulator E2F3, and the anti-apoptotic protein BCL2, and also causes induction of pro-apoptotic proteins including caspases. “In so doing, they collectively regulate cell proliferation and cell cycle, impact cell death, and enhance the chemosensitivity to 5-FU,” Ju explained. Seeing as the miRNA is lost as the colon cancer progresses, his lab sought to restore miR-129 activity. But with an added twist.
The bottleneck for nucleic acid-based therapeutics such as miRNA or small interfering RNA (siRNA) is the delivery because “in order for it to be internalized it has to be packaged with a delivery vehicle,” Ju said. His lab substituted all 9 miR-129 uracils with 5-FU to create Mimic-1, a modified miRNA that can be taken up by colon cancer cells vehicle-free. An added benefit of Mimic-1 is that the 5-FU appears to synergize with the miRNA: a 100 µM 5-FU concentration was able to eliminate only about 20% of the 5-FU-resistant cells mentioned above, but only 100 nM of Mimic-1 was needed to eliminate 80% of the cells. “Which is a 3-log lower magnitude than 5-FU alone, indicating it does have the potential to overcome drug resistance.”
In a tail-vein-injected metastatic colon cancer model they showed that after two weeks of systemic miR-129 mimic injection the metastases were eliminated in vivo. No toxicity was associated with the treatment.
Platform technology
Ju sees the 5-FU miRNA mimics as a platform technology. The combination of low concentration needed to see high efficacy, coupled with the vehicle-free delivery, will allow the platform to get around some of the toxicity issues connected with packaging materials in recent trials.
And it delivers a “cluster bomb because the miRNA itself is multitargeted—we really need the multitargeted entity to get rid of these tumor cells, because they’re notoriously resistant,” he emphasizes. “We want to hit them all at the same time, and you cannot do that with combined small molecule drugs” because of the associated severe toxicity.
Ju ended his presentation showing that miRNAs are stable enough, and predictive enough, to have potential as a prognostic biomarker for colon cancer.