Abstract Disturbed cholesterol homeostasis plays critical roles in the development of multiple diseases, such as cardiovascular diseases (CVD), neurodegenerative diseases and cancers, particularly the CVD in which the accumulation of lipids (mainly the cholesteryl esters) within macrophage/foam cells underneath the endothelial layer drives the formation of atherosclerotic lesions eventually. More and more studies have shown that lowering cholesterol level, especially low-density lipoprotein cholesterol level, protects cardiovascular system and prevents cardiovascular events effectively. Maintaining cholesterol homeostasis is determined by cholesterol biosynthesis, uptake, efflux, transport, storage, utilization, and/or excretion. All the processes should be precisely controlled by the multiple regulatory pathways. Based on the regulation of cholesterol homeostasis, many interventions have been developed to lower cholesterol by inhibiting cholesterol biosynthesis and uptake or enhancing cholesterol utilization and excretion. Herein, we summarize the historical review and research events, the current understandings of the molecular pathways playing key roles in regulating cholesterol homeostasis, and the cholesterol-lowering interventions in clinics or in preclinical studies as well as new cholesterol-lowering targets and their clinical advances. More importantly, we review and discuss the benefits of those interventions for the treatment of multiple diseases including atherosclerotic cardiovascular diseases, obesity, diabetes, nonalcoholic fatty liver disease, cancer, neurodegenerative diseases, osteoporosis and virus infection. 

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Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics

Malignant melanoma is a type of very dangerous skin cancer. Histone modifiers usually become dysregulated during the process of carcinoma development, thus there is potential for a histone modifier inhibitor as a useful drug for cancer therapy. There is a multitude of evidence regarding the role of G9a, a histone methyltransferase (HMTase), in tumorigenesis. In this study, we first showed that G9a was significantly upregulated in melanoma patients. Using the TCGA database, we found a significantly higher expression of G9a in primary melanoma samples (n = 461) compared to normal skin samples (n = 551). Next, we knocked down G9a in human M14 and A375 melanoma cell lines in vitro via small interfering RNA (siRNA). This resulted in a significant decrease in cell viability, migration and invasion, and an increase in cell apoptosis. UNC0642 is a small molecule inhibitor of G9a that demonstrates minimal cell toxicity and good in vivo pharmacokinetic characteristics. We investigated the role of UNC0642 in melanoma cells, and detected its anti-cancer effects in vitro and in vivo. Next, we treated cells with UNC0642, and observed a significant decrease in cell viability in M14 and A375 cell lines. Furthermore, treatment with UNC0642 resulted in increased apoptosis. In immunocompetent mice bearing A375 engrafts, treatment with UNC0642 inhibited tumor growth. Results of Western blot analysis revealed that administration of UNC0642 or silencing of G9a expression by siRNA reduced Notch1 expression significantly and decreased the level of Hes1 in A375. All in all, the data from our study demonstrates potential of G9a as a therapeutic target in the treatment of melanoma.

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Abnormal overexpression of G9a in melanoma cells promotes cancer progression via upregulation of the Notch1 signaling pathway.

The use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) brings remarkable benefits for the survival of patients with advanced NSCLC harboring EGFR mutations. Unfortunately, acquired resistance seems to be inevitable and limits the application of EGFR-TKIs in clinical practice. This study reported a common molecular mechanism sustaining resistance and potential treatment options to overcome EGFR-TKIs resistance.EGFR-TKIs resistant NSCLC cells were established and confirmed by MTT assay. Cholesterol content was detected and the promotional function of cholesterol on NSCLC growth was determined in vivo. Then, we identified ERRα expression as the downstream factor of cholesterol-mediated drug resistance. To dissect the regulatory mechanism, we conducted experiments, including immunofluorescence, co-immunoprecipitation, luciferase reporter assay and chromatin immunoprecipitation assay.Long-term exposure to EGFR-TKIs generate drug resistance with the characteristic of cholesterol accumulation in lipid rafts, which promotes EGFR and Src to interact and lead EGFR/Src/Erk signaling reactivation-mediated SP1 nuclear translocation and ERRα re-expression. Further investigation identifies ERRα as a target gene of SP1. Functionally, re-expression of ERRα sustains cell proliferation by regulating ROS detoxification process. Lovastatin, a drug used to decrease cholesterol level, and XCT790, an inverse agonist of ERRα, overcome gefitinib and osimertinib resistance both in vitro and in vivo.Our study indicates that cholesterol/EGFR/Src/Erk/SP1 axis-induced ERRα re-expression promotes survival of gefitinib and osimertinib-resistant cancer cells. Besides, we demonstrate the potential of lowing cholesterol and downregulation of ERRα as effective adjuvant treatment of NSCLC. 

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Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERRα re-expression

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As an essential lipid, cholesterol is of great value in keeping cell homeostasis, being the precursor of bile acid and steroid hormones, and stabilizing membrane lipid rafts. As a kind of cholesterol metabolite produced by enzymatic or radical process, oxysterols have drawn much attention in the last decades. Among which, the role of 25-hydroxycholesterol (25-HC) in cholesterol and bile acid metabolism, antivirus process, and inflammatory response has been largely disclosed. This review is aimed at revealing these functions and underlying mechanisms of 25-HC.

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Multiple Roles of 25-Hydroxycholesterol in Lipid Metabolism, Antivirus Process, Inflammatory Response, and Cell Survival.

Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer. Besides genetic and environmental factors, epigenetic alterations contribute to the tumorigenesis of NSCLC. Epigenetic changes are considered key drivers of cancer initiation and progression, and altered expression and activity of epigenetic modifiers reshape the epigenetic landscape in cancer cells. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2) is a histone methyltransferase and catalyzes mono- and di-methylation at histone H3 lysine 9 (H3K9me1 and H3K9me2, respectively), leading to gene silencing. EHMT2 overexpression has been reported in various types of cancer, including ovarian cancer and neuroblastoma, in relation to cell proliferation and metastasis. However, its role in NSCLC is not fully understood. In this study, we showed that EHMT2 gene expression was higher in NSCLC than normal lung tissue based on publicly available data. Inhibition of EHMT2 by BIX01294 (BIX) reduced cell viability of NSCLC cell lines via induction of autophagy. Through RNA sequencing analysis, we found that EHMT2 inhibition significantly affected the cholesterol biosynthesis pathway. BIX treatment directly induced the expression of SREBF2, which is a master regulator of cholesterol biosynthesis, by lowering H3K9me1 and H3K9me2 at the promoter. Treatment of a cholesterol biosynthesis inhibitor, 25-hydroxycholesterol (25-HC), partially recovered BIX-induced cell death by attenuating autophagy. Our data demonstrated that EHMT2 inhibition effectively induced cell death in NSCLC cells through altering cholesterol metabolism-dependent autophagy.

https://www.mdpi.com/1422-0067/21/3/1002/pdf

EHMT2 Inhibition Induces Cell Death in Human Non-Small Cell Lung Cancer by Altering the Cholesterol Biosynthesis Pathway.

Objectives
N6-Methyladenosine (m6A) is the most prevalent mRNA modification, which modulates mRNA export, splicing, and stability. Recent studies showed that m6A is involved in cell differentiation such as spermatogenesis, but the underlying mechanism is still unclear. This study was aimed to investigate whether alkylation repair homolog 5 (ALKBH5)-dependent m6A demethylation plays a role in metabolic-related cell differentiation.


Methods
Expression analysis upon differentiation was done using R studio on publicly available data from two independent cell differentiation models, adipogenic 3T3-L1 and myogenic C2C12. M6A levels in mRNA were measured using dot blot. Knockdown of ALKBH5 was conducted by small interference RNA at two days prior to differentiation induction. For phenotypic measures upon differentiation, staining analysis by Giemsa and Oil Red-O was conducted for 3T3-L1 and C2C12 cells, respectively, followed by histogram analysis using ImageJ. Total RNA was isolated from cells and was reverse-transcribed to cDNA, followed by qRT-PCR for gene expression analysis.


Results
Comparative analysis of adipogenesis and myogenesis models revealed common pathways, including oxidative phosphorylation. Among a-ketoglutarate-dependent demethylases, Alkbh5 expression was substantial and its substrate, m6A level was changed upon induction of differentiation. Staining analysis showed that Alkbh5 knockdown using siRNA significantly increased production of triglycerides and myotubes in 3T3-L1 and C2C12, respectively, indicating promotion of cell differentiation. To identify putative targets, expression data using microarray and qRT-PCR were analyzed. Expression of CEBPb and myogenin among adipogenic and myogenic markers was significantly altered upon Alkbh5 knockdown in 3T3-L1 and C2C12, respectively.


Conclusions
The results together showed that ALKBH repression promoted cell differentiation in both adipogenic and myogenic models, possibly regulating early differentiating markers such as CEBPb and myogenin, respectively.


Funding Sources
NRF of Korea (2018R1D1A1B07051274 & 22A20130012143).

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Alkbh5, a RNA Demethylase, Is Involved in Fine-tuning of Cell Differentiation (FS11-07-19)

Objectives
G9A is a eukaryotic histone methyltransferase that contributes to transcriptional silencing of tumor suppressor genes by modulating histone 3 lysine 9 methylation (H3K9m). It has been recently suggested as a promising therapeutic target for breast cancer and neuroblastoma. This study was aimed to investigate the effect of G9A inhibition and its cellular metabolic mechanisms.


Methods
Using OncomineTM, G9A overexpression in lung cancer was assessed. Cell viability upon treatment of G9A inhibitor (BIX01294, BIX) and siG9A was measured by MTT and IncuCyteR assays. Additionally, apoptosis and autophagy were analyzed through western blots. In order to identify targets, transcriptomes using RNA sequencing was conducted upon BIX treatment. Further functional relevance of targets was validated using Chromatin IP and recovery tests.


Results
BIX-mediated inhibition of G9A reduced cell viability of lung cancer cells via induction of autophagy. Through RNA sequencing, we found that G9A inhibition mainly affected cholesterol biosynthesis pathway. BIX directly induced the expression of SREBF2 gene, by lowering H3K9me1 and H3K9me2 at the promoter. A cholesterol biosynthesis inhibitor, 25-HC, partially recovered BIX-induced cell death by attenuating autophagy. Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death.


Conclusions
Our data suggests that cholesterol metabolism can be a potential therapeutic target by G9A inhibition and its induction of autophagic cell death.


Funding Sources
NRF of Korea grant (2018R1D1A1B07051274); BK21 Plus Project (22A20130012143).

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Seo Yoon Choi

Papers

EHMT2 Inhibition Induces Cell Death in Human Non-Small Cell Lung Cancer by Altering the Cholesterol Biosynthesis Pathway.

Alkbh5, a RNA Demethylase, Is Involved in Fine-tuning of Cell Differentiation (FS11-07-19)

Cholesterol Biosynthesis Is Required for Cell Death in Response to G9A Inhibitor in Lung Cancer (P05-010-19)