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Tumor suppressive microRNAs for cancer therapy

Periodic Reporting for period 1 - RxmiRcanceR (Tumor suppressive microRNAs for cancer therapy)

Reporting period: 2018-09-01 to 2020-02-29

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 (RFA), trans-arterial chemoembolization (TACE) and surgical resections. Both Sorafenib and Regorafenib improve survival only by a few months and have major side effects. This year (2020) a new combination therapy against HCC was developed using anti-PD-L1 monoclonal antibody and anti-VEGF-A increasing median survival by less than 3 months! Our program addresses this challenge of improving survival of patients with HCC.

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 propose 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; 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, expression of these will be 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. This simulates the “natural” augmentation of the immune system against cancer (the US FDA recently approved the CAR-T approach) that is currently driving the reality of cancer treatment. My program is to navigate, through a number of new technologies that I will develop, the “hormonal” effect of tumor suppressive miRs into an anti-cancer therapeutic platform. I plan to assess this new approach for HCC and, once the results are positive, the same could be applied to other tumor types (post-ERC research).

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 main theme for this ERC is that we could encounter the “hormonal” property of microRNAs that we had reported before in our past papers from the lab and upcoming revised manuscript (Gastroenterology 2016;151:999–1010; Gastroenterology 2017;153:1404–1415; Chofit et al A Novel ROR Agonist Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) progression by miR-122 Up-Regulation, under revision in Gastroenterology), for anti-hepatocellular carcinoma (HCC) therapy. We found that tumor suppressive microRNAs are secreted from one cell and are affecting a neighboring cell. The ERC program undertakes this phenomenon to be translated into a therapeutic approach. To this aim we have commenced parallel investigations including a high-through put screening and a bias targeted approach to identify the tumor suppressive microRNAs and identify how to up-regulate their expression. Herein are individual summaries describing the different parallel approaches.

Hepatocellular carcinoma (HCC) is one of the most common solid cancers worldwide. The CD24 cell surface molecule was found to be overexpressed in HCC tumor tissues. CD24 protein plays an important role in self-renewal, proliferation, migration, invasion and drug resistance of HCC. 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 accordance, we show that CD24 is a direct gene target of miR-122. In addition, in vitro experiments demonstrated that over-expression of miR-122 in a hepatoma cell line results in reduced levels of the endogenous Cd24 mRNA levels and inhibition of cell proliferation. In addition, we found a strong negative correlation between the levels of miR-122 and CD24 in a large set of HCC human samples. Furthermore, we show that in different stages of progression from cirrhotic human liver to pre-tumorigenic condition, CD24 is up-regulated whereas miR-122 is down-regulated, exhibiting inverse correlation. Our results reveal that miR-122 acts as a regulator of CD24. Thus, treatments that increases the miR-122 expression, could serve as an inhibitor of cancer cell proliferation (Figure 1).

Recently it was found that wild-type KRAS expression is increased in HCC compared with non-tumorous liver, which correlated with tumor size, proliferation and poor survival of patients (Dietrich 2018). This observation lead us to speculate that regulating KRAS expression in HCC could encounter a therapeutic effect. This is substantiated by the observation that sorafenib treatment, currently a standard treatment for HCC, caused a dose-dependent upregulation of KRAS in HCC cells, which was associated with the development of sorafenib resistance, and KRAS inhibition could resensitise those cells for sorafenib-induced toxicity. Based on this observation and its rational we have initiated a study to determine the preferred microRNA to be upregulated in an effort to downregulate KRAS in HCC.

In order to determine microRNAs that can be activated in the liver to target KRAS, microRNA sequencing data was obtained through the GEO database from a study that sequenced the microRNA transcriptome in liver tissue that was adjacent to HCC tumors from human patients (Wojcicka 2014). The top 50 microRNAs that were expressed in normal liver tissue were selected to be screened for possible seed sequences in the 3'UTR of KRAS using RNA-RNA hybridization prediction websites including 'Target Scan' and 'RNA Hybrid'. MicroRNAs which displayed seed sequences in the 3'UTR of human and mouse 3'UTR sequences were identified wherein any score below -25 Kcal/mol was considered for potential binding (Figure 2).

Next we wanted to confirm that the miRs of interest are indeed expressed by the liver. qPCR on hepatic RNA from human liver samples indicated that identified anti-KRAS miRs were significantly expressed in liver (Figure 2A). Furthermore transfection of different combinations of these miRs into the Panc1 cell line (wherein mutated KRAS acts as a driver oncogene), generally resulted in a trend for reduced KRAS at the protein level (Figure 3B), however more work is required to confirm their individual effects on KRAS, cell growth, and tumorigenesis in in vivo models (Figure 3).

Since our goal is to harness the ability of small molecule activators of transcription to induce upregulation and secretion of miRs from the liver (Chai 2017) in order to inhibit HCC, we next wanted to know whether we can upregulate specific anti-KRAS miRs via a common transcription factor binding site in their promoter. To test this we exposed Panc1 cells to the Esr1 modulator, Tamoxifen, for which we found binding sites in the promoter region of miR-143, miR-145, let-7g, and let-7c. Results showed a dose-dependent increase in the expression of miR-143, miR-145, and let-7c, but not let-7g (Figure 4).

miR-122 is a liver specific microRNA with well-known tumor suppressive properties. Over-expression of miR-122 is considered as a potential treatment for HCC. Retinoic Acid Receptor (RAR) related orphan receptor (ROR) is a transcription factor (TF) that binds to miR-122 promoter and induce its expression. Identifying small synthetic molecules that activates ROR will induce the endogenous expression of miR-122. 42 small compounds with high potential to activate ROR were obtained by computational high throughput screening performed together with Prof. Amiram Goldblum. The activity of candidate compounds (at concentration of 5M) was tested for growth inhibition of two human cell lines: Huh7 (HCC) and Capan1 (pancreatic metastasis in liver). Cell growth rate was examined using live imaging microscopy (Incucyte system) for three and a half days. This screening revealed five compounds that reduced Huh7 cell growth and four compounds that reduced Capan1 cell growth compared to DMSO treated control cells. Two compounds, RA1 and RA7 inhibited growth of both cell lines (Fig 4). Next, we continued with the analysis of the five compounds that inhibited Huh7 cells growth, using 4 different concentrations, 10M, 5M, 1M and 0.1M. Out of the five molecules, RA12 exhibited a dose-dependent activity on cell growth, highest inhibition at a concentration of 10M, whereas compound RA1 exhibited the highest inhibitory effect at 0.1M (Fig 5). We next examined the ability of these 2 compounds, at increasing concentrations, to enhance miR-122 promoter activity in Huh7 cells previously transfected with miR-122 promoter reporter plasmid expressing EGFP. Of the two compounds only RA1 induced miR-122 promoter activity (Fig 6). In parallel, we compared RA2 and RA3 to RA1 for miR-122 promoter activation and for mature miR-122 secretion from Huh7 cells. As seen in Fig X, RA2 exhibited no activity; RA3 enhanced miR-122 secretion, whereas RA1 enhanced both, promoter activity and secretion (Fig 5).
Hepatocellular carcinoma (HCC) is ranked as the third most common cause of cancer related mortality worldwide. During hepatocarcinogenesis, liver cells gain somatic mutations. Almost half of the human genome consists of Transposable Elements (TEs) sequences, the vast majority of these TEs are mutated and are not capable to transpose in the genome. In the human genome, an exception of the TEs is the LINE1 retrotransposons family, of which 80-100 copies kept the capability to transpose and induce genomic changes. The LINE1 retrotransposons family is a driving force for somatic mutations that were shown to be pathological in many diseases, including HCC. Recent studies shown that genetic changes as retrotransposition of LINE-1 retrotransposons are common feature of hepatocarcinogenesis and cancer progression. In this project, we aim to investigate regulation of the LINE1 retrotransposition and mobilization processes by miRNAs (miRs). First, as a supervised approach, we used computational predictions of miRs that target known LINE1 regulators. From the miRs list that were found to target LINE-1 regulators miRs with known oncogenic and tumor suppressive function in HCC were chosen for further investigation. We show that known oncogenic miR-222-3p and miR-93-5p have a positive effect on LINE-1 retrotransposition in different HCC cell lines in vitro, using RNAi and CRISPR technologies. These miRs show positive correlation between oncogenic function and enhanced retrotransposition.
Next, using CRISPR-cas9 screen as a non-supervised approach, we will generate knockouts in most miR genes in order to identify miRs that regulate LINE1, in a genome wide manner and further proof the correlation between oncogenic function and enhancing LINE-1 transposition.