Periodic Reporting for period 4 - RxmiRcanceR (Tumor suppressive microRNAs for cancer therapy)
Berichtszeitraum: 2023-03-01 bis 2024-08-31
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, today HCC is 3rd leading cause of death of cancer patients. 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, Regorafenib and immune check points inhibitors (ICI) combinations, improve survival only by a few months and have major side effects. Our program addresses this challenge of improving survival of patients with HCC with a novel therapeutic approach.
HCC is the 3rd leading cause of cancer related deaths. This causes a huge burden on the society. In addition of being mildly effective some compounds as ICI are expensive. This fact will refrain many patients in rural societies from therapy. 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.
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.
HCC is the 3rd leading cause of cancer related deaths. This causes a huge burden on the society. In addition of being mildly effective some compounds as ICI are expensive. This fact will refrain many patients in rural societies from therapy. 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.
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.
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). 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. By performing a high throughput screen, we have identified a number of compounds that increase expression as well as secretion. These are CDK5 inhibitors. Two of those are already in the clinic. Now we are conducting in vivo experiments to validate the anti-cancer effect of increasing miR-34a.
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, 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. To further validate these results, we have generated a miR-122 KO mice. Surprisingly we found that in this mouse, liver metastasis is progressing rapidly compared to wt mice. We have investigated this and found that this effects is dependent on ADAM17, a known target of miR-122. Upon knocking out miR-122 ADAM17 increase (as well as ADAM10, which has a similar effect). Furthermore, in a 2nd compound development screen we found additional 4 compounds with an even “better” effect of increasing expression and secretion of miR-122. The most effective compound is now investigated in vivo for anti-tumor effects. Surprisingly, we found that one of the compounds increases the hepatokine FGF21 levels. This finding could make it an anti-pancreatitis compound. Today, there is no available specific treatment for pancreatitis.
3. Determine the tumor suppressive mechanism of miR- 122. In this aim we have moved in a few directions:3a. Establish miR-122 knockout mice. 3b. 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, and screening for microRNAs regulating HCC cell proliferation. We have identify miR-222 that suppress retrotransposition. Now we are investigating what regulates this miR-222. We find that the effect of miR-222 is through miR-222 host gene non-coding RNA.
1. Identify and assess the activity of compounds that are increasing expression and secretion of the tumor suppressive miR-34a (microRNA – 34a). 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. By performing a high throughput screen, we have identified a number of compounds that increase expression as well as secretion. These are CDK5 inhibitors. Two of those are already in the clinic. Now we are conducting in vivo experiments to validate the anti-cancer effect of increasing miR-34a.
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, 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. To further validate these results, we have generated a miR-122 KO mice. Surprisingly we found that in this mouse, liver metastasis is progressing rapidly compared to wt mice. We have investigated this and found that this effects is dependent on ADAM17, a known target of miR-122. Upon knocking out miR-122 ADAM17 increase (as well as ADAM10, which has a similar effect). Furthermore, in a 2nd compound development screen we found additional 4 compounds with an even “better” effect of increasing expression and secretion of miR-122. The most effective compound is now investigated in vivo for anti-tumor effects. Surprisingly, we found that one of the compounds increases the hepatokine FGF21 levels. This finding could make it an anti-pancreatitis compound. Today, there is no available specific treatment for pancreatitis.
3. Determine the tumor suppressive mechanism of miR- 122. In this aim we have moved in a few directions:3a. Establish miR-122 knockout mice. 3b. 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, and screening for microRNAs regulating HCC cell proliferation. We have identify miR-222 that suppress retrotransposition. Now we are investigating what regulates this miR-222. We find that the effect of miR-222 is through miR-222 host gene non-coding RNA.
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. 4. Our new results show that miR-122 encounters anti-metastasis activity in the liver. 5. We prove that miR-122 regulates liver tolerance (!). This is a major finding in our minds. It means we could reduce liver tolerance in the liver and possible reject primary and metastatic tumors.