Periodic Reporting for period 3 - RxmiRcanceR (Tumor suppressive microRNAs for cancer therapy)
Periodo di rendicontazione: 2021-09-01 al 2023-02-28
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, 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. 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 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. We will then be able to select those that are less toxic and have a significant effect on tumor development and progression. Already for miR-34a we identified >6 compounds with an effect of increasing this miR. For miR-122 we found >4. Both are now tested for anti-tumor effects. We have asked an extension of one year to complete the in vivo anti-tumor experiments.
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.
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. 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 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. We will then be able to select those that are less toxic and have a significant effect on tumor development and progression. Already for miR-34a we identified >6 compounds with an effect of increasing this miR. For miR-122 we found >4. Both are now tested for anti-tumor effects. We have asked an extension of one year to complete the in vivo anti-tumor experiments.
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). 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. In addition, we have identified six compounds out of a chemical library with an effect of increasing expression and secretion of miR-34a. Furthermore, we also applied new compounds which increase secretion of miR’s. In in vitro studies we already see significant anti-tumor effects. Dose and kinetic studies in mice identified for us the preferred treatment protocol which is tested now.
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. In special CD24 manipulated organoids, in miR-122 KO mice and CD24 KO mice we proved that miR-122 suppress CD24 and as a result suppress liver ductular proliferation, a pre-malignant condition in all chronic liver diseases resulting in cirrhosis and hepatocellular carcinoma.
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.
5. Assess tumor suppressive effects of additional microRNAs: miR-21 and miR-9. 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. We developed a miR-9 small compounds screening system and now are ready for the identification of miR-9 small compound enhancer. Mir-9 is expected to have anti-liver fibrotic effects.
6. We had developed a miR-122 KO mice. The liver of the miR-122 KO is inflamed and fibrotic from 3 weeks of age. In the inflamed livers of these mice, there is an increase in Kupffer and T cells as well as an increase in liver damage markers – including fibrosis, ductular proliferation and apoptosis. Gene expression analysis demonstrates overexpression of TLR genes in miR-122 KO mice and enrichment of genes that are associated with TNF and NFB signaling. In microbiome depleted miR-122 KO mice, the liver damage was mildly decreased. Specific subsets of T cells were analyzed by FACS and we found that in effector CD8 cells, immune checkpoints are downregulated. In a preliminary experiment where CD8 cells were depleted, we observed no change in liver apoptosis levels. Additionally, we observed an increase in the gene expression of SAA which could drive pathogenic Th17 cell differentiation. Protein levels of IL-17, which is secreted from Th17 cells, is increased in the livers of miR-122 KO mice. Comparing RNAseq data of these mice with humans with AIH showed a significant overlap. In AIH patients an anti-correlation between levels of miR-122 in the liver and inflammation activity index was observed. Specifically, TLR signaling, NFB signaling and Th17 signaling pathways are all significantly differentially expressed in AIH patients with high inflammation activity index as in the miR-122 KO mice. We hypothesize that the immune response exhibited in the miR-122 KO mice is a result of a dysregulated response to the natural microbiome in the absence of miR-122. Overexpression of TLRs could lead to excessive NFB activation and TNF signaling. Further analysis is required in order to determine the pathways and specific cellular responses involved in regulation of the liver immune tolerance by miR-122.
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. In addition, we have identified six compounds out of a chemical library with an effect of increasing expression and secretion of miR-34a. Furthermore, we also applied new compounds which increase secretion of miR’s. In in vitro studies we already see significant anti-tumor effects. Dose and kinetic studies in mice identified for us the preferred treatment protocol which is tested now.
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. In special CD24 manipulated organoids, in miR-122 KO mice and CD24 KO mice we proved that miR-122 suppress CD24 and as a result suppress liver ductular proliferation, a pre-malignant condition in all chronic liver diseases resulting in cirrhosis and hepatocellular carcinoma.
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.
5. Assess tumor suppressive effects of additional microRNAs: miR-21 and miR-9. 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. We developed a miR-9 small compounds screening system and now are ready for the identification of miR-9 small compound enhancer. Mir-9 is expected to have anti-liver fibrotic effects.
6. We had developed a miR-122 KO mice. The liver of the miR-122 KO is inflamed and fibrotic from 3 weeks of age. In the inflamed livers of these mice, there is an increase in Kupffer and T cells as well as an increase in liver damage markers – including fibrosis, ductular proliferation and apoptosis. Gene expression analysis demonstrates overexpression of TLR genes in miR-122 KO mice and enrichment of genes that are associated with TNF and NFB signaling. In microbiome depleted miR-122 KO mice, the liver damage was mildly decreased. Specific subsets of T cells were analyzed by FACS and we found that in effector CD8 cells, immune checkpoints are downregulated. In a preliminary experiment where CD8 cells were depleted, we observed no change in liver apoptosis levels. Additionally, we observed an increase in the gene expression of SAA which could drive pathogenic Th17 cell differentiation. Protein levels of IL-17, which is secreted from Th17 cells, is increased in the livers of miR-122 KO mice. Comparing RNAseq data of these mice with humans with AIH showed a significant overlap. In AIH patients an anti-correlation between levels of miR-122 in the liver and inflammation activity index was observed. Specifically, TLR signaling, NFB signaling and Th17 signaling pathways are all significantly differentially expressed in AIH patients with high inflammation activity index as in the miR-122 KO mice. We hypothesize that the immune response exhibited in the miR-122 KO mice is a result of a dysregulated response to the natural microbiome in the absence of miR-122. Overexpression of TLRs could lead to excessive NFB activation and TNF signaling. Further analysis is required in order to determine the pathways and specific cellular responses involved in regulation of the liver immune tolerance by miR-122.
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 (!) (section 6 in the above paragraph). 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.
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-vivo experimentations.
2. The tumor suppressive effects of miR-122 on HCC and metastasis is already progressing both in in-vitro and in-vivo experimentation.
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.
4. We can manipulate liver tolerance for metastasis and improve survival by suppressing liver metastasis growth.
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-vivo experimentations.
2. The tumor suppressive effects of miR-122 on HCC and metastasis is already progressing both in in-vitro and in-vivo experimentation.
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.
4. We can manipulate liver tolerance for metastasis and improve survival by suppressing liver metastasis growth.