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Tumor suppressive microRNAs for cancer therapy

Periodic Reporting for period 2 - RxmiRcanceR (Tumor suppressive microRNAs for cancer therapy)

Reporting period: 2020-03-01 to 2021-08-31

What is the problem/issues being addressed?:
Cancer is a leading cause of morbidity and mortality but our current therapeutic tool box for many cancers is quite empty. Although hepatocellular carcinoma (HCC) incidence is increasingly causing mortality, the current therapeutic options are over 10 years old and are mostly insufficient. They are mainly directed for focal/local HCCs and include liver transplantation, radiofrequency ablation, trans-arterial chemoembolization and surgical resections. Both Sorafenib and Regorafenib improve survival only by a few months and have major side effects. This year a new combination therapy against HCC was developed increasing median survival by less than 3 months! Our program addresses this challenge of improving survival of patients with HCC with a novel therapeutic approach. Our special approach is based on our concept that microRNAs serve as “hormones”: produced in one cell/organ, and delivered in special vehicles, reaching remote cells and organs. Some microRNAs are tumor suppressors as miR-34 and miR-122. Our program is aimed 1st to identify additional microRNAs with tumor suppression activity, 2nd regulate/increase expression and secretion of known and newly found tumor suppressive microRNAs with “small” compounds. And 3rd after the identification of these compounds to assess their anti-tumor activity in vitro and in vivo (until today we have made progress in all three fronts).

Why is it important for the society?:
HCC is the 3rd leading cause of cancer related deaths. This causes a huge burden on the society. In our approach we expect to identify small compounds to be developed into small drugs against HCC. We expect to encounter less side effects due to the fact that we are “harnessing” natural regulatory machinery, that regulates tumor suppressive microRNAs expression and only enhances their activity. Furthermore, our multimodality approach will enable the development of many compounds. We will then be able to select those that are less toxic and have a significant effect on tumor development and progression.

What are the overall objectives?:
Two parallel revolutions are concurrently developing in biomedical research, which are relevant to our grant program; one is the understanding that mammalian cells regulate their gene expression and modify their phenotype also by microRNAs. The second is the fact that these mammalian cells shed different sizes of micro-vesicles that serve as vehicles for microRNAs as well as for proteins. My hypothesis is that by artificially increasing the biogenesis and secretion of microRNAs, with tumor suppressive properties, and enveloped in “natural” vehicles, e.g. exosomes, and secreted out of cells to transduce local and distant cancer cells as well as metastasis, thereby causing tumor regression and improving rate of survival. This “natural” path is also expected to have fewer side effects. In my team work we have already shown that miRs could function as hormones. Here, I wish to harness the inherent human tissue microRNA-based anti-tumor activity for HCC therapy, by enhancing this “natural” intra and inter-cellular property. My overall objective is to develop an entirely new anti-cancer therapeutic platform for HCC by increasing the expression of tumor suppressive miRs and inducing the delivery of these miRs to tumors to cause tumor regression and increase survival rates.
Our activity runs in parallel in a number of direction to reach as securely and with the highest probability as possible our ultimate goal/objective: To facilitate the tumor-suppressive microRNAs hormonal properties in vivo in animal models with newly identified small compounds. This activity is layered through the following activities/aims:
1. Identify and assess the activity of compounds that are increasing expression and secretion of the tumor suppressive miR-34a (microRNA – 34a) (see figure 1). MiR-34a is known as a tumor suppressive microRNA. Our program in this ERC project is to increase its expression, induce its secretion from cells, preferentially not from tumor cells to have an anti-tumor effect against the surrounding cancer cells. Whether these are primary tumors developing or metastasis.
2. Identify and assess the activity of compounds increasing expression and secretion of miR-122. We had reported before in our past papers (Gastroenterology 2016;151:999–1010; Gastroenterology 2017;153:1404–1415), that miR-122 is regulated by specific transcription factors. One specific compound, RS2982 (see figure 3 in the 2020 paper), that activates ROR, was found to be most effective. The anti-HCC activity of miR-122 is further investigated, to identify targets and mechanisms, see other sections of this report.
3. Determine the tumor suppressive mechanism of miR- 122 (see figure 2). In this aim we have moved in a few directions: 3a (see figure 2a). Establish miR-122 knockout mice. 3b (see figure 2b). CD24 is a cancer stem cell marker, also for primary liver cancers. CD24 signaling induces cancer cells proliferation. Our bioinformatic analysis revealed CD24 as a putative target gene of the well-known liver specific miR-122. It has been shown that the levels of miR-122 are drastically reduced in HCC. Since miR-122, a liver-specific tumor suppressor microRNA, and, CD24 have reciprocal expression in livers, we suggested that miR-122 could be a regulator of CD24.
4. Identify in a bias and an unbiased approaches microRNAs which regulate retrotransposition, a tumor initiating and progression events (figure 3a-c), and screening for microRNAs regulating HCC cell proliferation (figure 3d-e).
5. Assess tumor suppressive effects of additional microRNAs: miR-21 and miR-9 (see figure 4). 5a – miR-21 is a tumor promoting microRNA. We show data in this direction and investigated the mechanism. 5b – Targeting fibrosis, a pro-HCC factor: We show that increasing miR-9 is a potential therapeutic approach against HCC.
This program encounters numerous advantages over current anti-HCC approaches, and these include: 1. Regulating microRNAs by small compounds which are either already drugs approved or in the pipeline, saves time and funding. 2. MicroRNAs, which are the therapeutic effectors, are all natural produced/expressed in humans and then secreted. In particular tumor suppressive microRNAs as miR-122, miR-34a and also the anti-fibrotic microRNAs miR-9 and miR-21 are all expressed in the normal human livers. We increase their expression by activating transcription factors responsible for their expression. 3. We will assess combination treatments with activators of tumor suppressive microRNAs expression. 4. With nucleic acids, delivery is a paramount issue. By increasing the expression of microRNAs which than leads to microRNAs secretion we overcome a major hurdle in proposing a new therapeutic approach for small RNAs.
Expected results:
1. Now that we obtain the activators of expression and secretion of miR-34, following the high through – put screening, we are ready for in-vitro and in-vivo experimentations.
2. The tumor suppressive effects of miR-122 on HCC and metastasis is already progressing both in in-vitro and in-vivo experimentations (as we reported herein).
3. We have identified microRNAs which enhance LINE-1 retrotransposition. We are now progressing to in-vitro and in-vivo investigations once we have identified the microRNA’s to be targeted.
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