# ROS Signaling-Mediated Novel Biological Targets: Brf1 and RNA Pol III Genes

Authors: Liling Zheng; Yongluan Lin; Shuping Zhong
Journal: Oxidative Medicine and Cellular Longevity
 (2021)

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## Abstract

Biomolecule metabolism produces ROS (reactive oxygen species) under physiological and pathophysiological conditions. Dietary factors (alcohol) and carcinogens (EGF, DEN, and MNNG) also induce the release of ROS. ROS often causes cell stress and tissue injury, eventually resulting in disorders or diseases of the body through different signaling pathways. Normal metabolism of protein is critically important to maintain cellular function and body health. Brf1 (transcript factor II B-related factor 1) and its target genes, RNA Pol III genes (RNA polymerase III-dependent genes), control the process of protein synthesis. Studies have demonstrated that the deregulation of Brf1 and its target genes is tightly linked to cell proliferation, cell transformation, tumor development, and human cancers, while alcohol, EGF, DEN, and MNNG are able to induce the deregulation of these genes through different signaling pathways. Therefore, it is very important to emphasize the roles of these signaling events mediating the processes of Brf1 and RNA Pol III gene transcription. In the present paper, we mainly summarize our studies on signaling events which mediate the deregulation of these genes in the past dozen years. These studies indicate that Brf1 and RNA Pol III genes are novel biological targets of ROS.


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## Body

## 1. Introduction

Under physiological and pathophysiological conditions, the synthesis and metabolism of biomoleculesin vivo often produce ROS (reactive oxygen species), which reversibly or irreversibly induces cellular stress. This may further affect cell growth, proliferation and transformation and even lead to tumor development. These vital processes in cells are completed through different signaling pathways. Protein synthesis is critically important for normal cell growth. The synthesis of large quantities of proteins is required for tumor cell growth. Brf1 (transcription factor II-B related factor 1) is a key transcription factor, which specifically regulates RNA Pol III gene (RNA polymerase III-dependent gene) transcription. Pol III genes include tRNAs, 5S rRNA, and other small noncoding RNAs, such as 7SL RNA and U6 [1–3]. These Pol III genes are regulated by their transcription machineries. The transcriptional machinery complexes of tRNA genes include RNA polymerase III, TFIIIB, and TFIIIC, while those of 5S rRNA comprise RNA polymerase III and complexes of TFIIIA, TFIIIB, and TFIIIC. The TFIIIB complex consists of TBP, Brf1, and Bdp1. Studies have demonstrated that TFIIIB is associated with cell transformation and tumor development. Both oncogenes and tumor suppressors affect TFIIIB activity [1–3]. Brf1 and Pol III genes directly take part in the process of protein synthesis. Deregulation of Brf1 and Pol III genes is tightly linked to cell proliferation, cell transformation, and tumor development [4–9]. Brf1 is overexpressed in several human cancers, such as hepatocellular carcinoma, breast cancer, gastric cancer, prostate cancer, and lung cancer [5, 8, 10–13], while carcinogens, such as EGF (epidermal growth factor), DEN (Diethylnitrosamine), and MNNG (N-methyl-N′-nitro-N-nitrosoguanidine), and dietary factors (alcohol) induce the deregulation of Brf1 and Pol III genes [4, 7, 10, 13, 14]. This shows that the deregulation of Brf1 and Pol III genes plays a critical role in tumor development. The deregulation of Brf1 and Pol III genes is mediated by different signaling pathways. Our studies and others have indicated that MAPK (mitogen-activated protein kinase) subfamily members (ERKs, p38 kinases, and JNKs), MSK1 (mitogen- and stress-activated protein kinase 1), PLK1 (polo-like kinase 1, also called serine/threonine-protein kinases), and AMPK (5′ AMP-activated protein kinase or 5′ adenosine monophosphate-activated protein kinase) pathways involve the modulation of the activities of Brf1 and Pol III genes [4–7, 10, 11]. Therefore, investigating the signaling pathways which mediate the deregulation of these genes and identifying specific inhibitors may provide potential approaches for the treatments of human cancers.Back in 2011, alcohol has been classified as a carcinogen to humans by IACR (the International Agency for Research on Cancer) [15]. Subsequently, emerging studies indicate that alcohol consumption is associated with human cancers, which include hepatocellular carcinoma, breast cancer, gastric cancer, esophageal carcinoma, neck and head cancer, and others [5, 11, 16–19]. Alcohol consumption is a well-established risk factor for human cancers. Increasing the amounts of alcohol intake enhances the risk of cancer development. Chronic alcohol consumption results in the production of acetaldehyde and induction of CYP2E1 (cytochrome P450 2E1). Acetaldehyde is a by-product of alcohol metabolism catalyzed by ADH (alcohol dehydrogenase). Acetaldehyde has direct effects on mutagenesis and carcinogenesis in vivo and in vitro [1, 2, 12], whereas CYP2E1 is associated with the release of ROS (reactive oxygen species). CYP2E1 takes part in the conversion of procarcinogens to carcinogens. Alcohol intake augments the cellular production of ROS to cause cell stress, eventually resulting in diseases with cell and tissue injury. Our studies have demonstrated that alcohol treatment in various cells caused different alterations of TFIIIB subunits. The changes result in the deregulation of Brf1 and Pol III genes through different signaling molecules to promote cell proliferation, cell transformation, and tumor development [4, 5, 7, 8]. Except for alcohol, many carcinogens also induce ROS release through different pathways in cells and tissues. As the deregulation of Brf1 and Pol III genes is tightly associated with cell proliferation, cell transformation, and cancer development, summarizing these signaling events and analyzing the underlying molecular mechanisms will benefit developing specific inhibitors to prevent cancer development in humans.


## 2. Alcohol-Induced Deregulation of Brf1 and Pol III Genes and Breast Cancer

Breast cancer is the most frequent cancer in women in the United States. Over 496,000 new cases have been diagnosed every year in the country. Early studies have indicated that alcohol consumption is associated with the risk of breast cancer [20, 21]. Emerging studies have clearly demonstrated that alcohol consumption is an established risk factor for breast cancer [22–25]. Approximately 80% of all cases of breast cancer are ER+ (estrogen receptor-positive) and 20% ER- (estrogen receptor-negative) [1, 2, 10]. Alcohol intake is associated with ER+ cases of breast cancer more than ER- cases [1]. This implies that ERα may play an important role in alcohol-associated breast cancer.


### 2.1. ERα Mediates Alcohol-Induced Transcription of Brf1 and Pol III Genes

Our study has demonstrated that alcohol treatment of breast cancer cells enhances the activity of the ERα promoter and increases the cellular levels of ERα mRNA and protein [4], while alcohol-induced RNA Pol III gene transcription in ER+ breast cancer cell lines is much higher than that in ER- cells [4, 5]. This insinuates that ERα may mediate alcohol-induced Brf1 expression, which is a specific transcription factor of RNA Pol III genes to control the transcription of these genes. Inhibition of ERα decreases the transcription of Brf1 and Pol III genes, while by enhancing ERα activity by its ligand, E2 (17β-estradiol) augments the expression of these genes [4]. Tam (tamoxifen) is an antagonist of ER in breast tissue, which competitively binds to ER to reduce its activity. Tam is used to routinely treat patients of both early and advanced ER+ breast cancer in women [26]. Interestingly, Tam represses Brf1 expression and Pol III gene transcription [6]. Repressing ERα by its siRNA or Tam reduces Brf1 expression and Pol III gene transcription, leading to a reduction of the rates of alcohol-increased cell proliferation and colony formation [4–6]. These studies demonstrate that ERα modulates alcohol-induced deregulation of Brf1 and Pol III genes, which is tightly linked to cell transformation and tumor development [4–6].


### 2.2. . Runx2 Participates in the Regulation of Brf1 and Pol III Genes

Runx2 (Runt-related transcription factor 2) is a downstream component of the ERα pathway. Runx2 is a transcription factor which is associated with osteoblast differentiation [27] and has been described as an oncogene [28, 29]. Runx2 is related to mammary gland development and ER+ breast cancer [28]. A high level of Runx2 is found in breast cancer cell lines [29]. Runx2 controls the expression of genes which are associated with tumor cell growth and migration [30]. Both E2 (17-β estradiol) and ERα upregulate Runx2 transcription [31, 32], whereas Runx2 can bind to the promoter of ERα to enhance its expression. It indicates that Runx2 not only is regulated by ERα but also can modulate ERα expression. A recent study indicates that alcohol increases Runx2 transcription in ER+ breast cancer cells [7]. Overexpressing Runx2 by its constructs or repressing Runx2 expression by its siRNA augments or attenuates alcohol-induced transcription of Brf1 and Pol III genes [7]. Reduction of ERα activity weakens alcohol-induced Runx2 transcription, leading to decreases in Brf1 and Pol III gene transcription [7]. Brf1 is overexpressed in most cases of ER+ breast cancer [5]. Overexpression of Brf1 is accompanied by high levels of ERα and Runx2 expression in these cases [5, 7]. High levels of Brf1 expression in ER+ breast cancer cases reveal a longer survival period after Tam treatment [5]. It is consistent with Tam-repressed Brf1 expression and Pol III gene transcription discussed above [6]. The actual levels of Brf1 expression of these patients after Tam treatment are reduced. Thus, their survival times are extended. It shows that the level of Brf1 expression in ER+ breast cancer cases can be used as a biomarker of diagnosis and prognosis of this disease.


## 2.1. ERα Mediates Alcohol-Induced Transcription of Brf1 and Pol III Genes

Our study has demonstrated that alcohol treatment of breast cancer cells enhances the activity of the ERα promoter and increases the cellular levels of ERα mRNA and protein [4], while alcohol-induced RNA Pol III gene transcription in ER+ breast cancer cell lines is much higher than that in ER- cells [4, 5]. This insinuates that ERα may mediate alcohol-induced Brf1 expression, which is a specific transcription factor of RNA Pol III genes to control the transcription of these genes. Inhibition of ERα decreases the transcription of Brf1 and Pol III genes, while by enhancing ERα activity by its ligand, E2 (17β-estradiol) augments the expression of these genes [4]. Tam (tamoxifen) is an antagonist of ER in breast tissue, which competitively binds to ER to reduce its activity. Tam is used to routinely treat patients of both early and advanced ER+ breast cancer in women [26]. Interestingly, Tam represses Brf1 expression and Pol III gene transcription [6]. Repressing ERα by its siRNA or Tam reduces Brf1 expression and Pol III gene transcription, leading to a reduction of the rates of alcohol-increased cell proliferation and colony formation [4–6]. These studies demonstrate that ERα modulates alcohol-induced deregulation of Brf1 and Pol III genes, which is tightly linked to cell transformation and tumor development [4–6].


## 2.2. . Runx2 Participates in the Regulation of Brf1 and Pol III Genes

Runx2 (Runt-related transcription factor 2) is a downstream component of the ERα pathway. Runx2 is a transcription factor which is associated with osteoblast differentiation [27] and has been described as an oncogene [28, 29]. Runx2 is related to mammary gland development and ER+ breast cancer [28]. A high level of Runx2 is found in breast cancer cell lines [29]. Runx2 controls the expression of genes which are associated with tumor cell growth and migration [30]. Both E2 (17-β estradiol) and ERα upregulate Runx2 transcription [31, 32], whereas Runx2 can bind to the promoter of ERα to enhance its expression. It indicates that Runx2 not only is regulated by ERα but also can modulate ERα expression. A recent study indicates that alcohol increases Runx2 transcription in ER+ breast cancer cells [7]. Overexpressing Runx2 by its constructs or repressing Runx2 expression by its siRNA augments or attenuates alcohol-induced transcription of Brf1 and Pol III genes [7]. Reduction of ERα activity weakens alcohol-induced Runx2 transcription, leading to decreases in Brf1 and Pol III gene transcription [7]. Brf1 is overexpressed in most cases of ER+ breast cancer [5]. Overexpression of Brf1 is accompanied by high levels of ERα and Runx2 expression in these cases [5, 7]. High levels of Brf1 expression in ER+ breast cancer cases reveal a longer survival period after Tam treatment [5]. It is consistent with Tam-repressed Brf1 expression and Pol III gene transcription discussed above [6]. The actual levels of Brf1 expression of these patients after Tam treatment are reduced. Thus, their survival times are extended. It shows that the level of Brf1 expression in ER+ breast cancer cases can be used as a biomarker of diagnosis and prognosis of this disease.


## 3. MAPKs Mediate Noncoding RNAs, Pol I and Pol III Gene Transcription

MAPKs (mitogen-activated protein kinases) are a family which includes three subfamilies: ERKs, p38 kinases, and JNKs. MAPKs are specific for phosphorylation of serine and threonine of protein amino acid residuals. MAPKs are involved in directing cellular responses, such as mitogen, stress, and heat shock. MAPKs play critical roles in cell proliferation, apoptosis, cell survival, gene expression, differentiation, cell transformation, and tumor development [33]. Our studies have indicated that MAPKs mediate Brf1 expression and RNA Pol III gene transcription.


### 3.1. MAPKs Mediate RNA Pol I- and Pol III-Dependent Gene Transcription through TBP

EGF is a ligand of EGFR (epidermal growth factor receptor). EGF increases TBP (TATA-binding protein) expression [34]. The latter is a general transcription factor, which participates in the regulation of RNA Pol I, Pol II, and Pol III gene transcription. EGF enhances TBP expression to upregulate RNA Pol I and Pol III gene transcription [34]. Mutated Ras decreases EGF-induced TBP promoter activity while blocking the EGFR pathway inhibits TBP expression [34, 35]. Repressing TBP expression results in a decrease in EGF-induced Pol I and Pol III gene transcription [34]. EGF induces phosphorylation of MAP kinases (ERKS, p38 kinases, and JNKs), whereas their chemical inhibitors (U0126, SB202910, and SP600125) of ERKs, p38 kinases, and JNKs inhibit EGF-induced TBP promoter activity, respectively [34]. In addition, expressing dominant-negative mutant forms of ERK2, p38, or JNK1 also block TBP promoter activity [34]. The further studies by different long fragments of the TBP promoter show that EGF-increased activity of the TBP promoter region targets the site of Ets through the MAP kinase pathway [34]. Once Ets is mutated, EGF lost the role in enhancement in the TBP promoter activity [34]. These studies clearly indicate that all of the three subfamilies of MAPKs are involved in EGF-induced TBP expression and RNA Pol I and RNA Pol III gene transcription. EGF-induced transcriptions of these genes are through the EGFR-Ras-MAPK pathway.


### 3.2. JNK1 and JNK2 Differently Modulate Brf1 Expression and Pol III Gene Transcription

JNKs are a subfamily of MAPKs, which include the three members of this subfamily: JNK1, JNK2, and JNK3, which are encoded byjnk1, jnk2, and jnk3 genes, respectively, to produce multiple isoforms through different splicing [36–38]. JNK1 and JNK2 are universally expressed in all organs, but JNK3 is expressed in a few tissues, such as the heart, testis, and brain [37]. JNKs are activated by stress, inflammation, heat shock, carcinogens, and mitogens [38, 39]. Stimulators activate JNKs, namely, phosphorylated JNKs which participate in complex cellular processes, such as inflammation, cell mitogenesis, apoptosis, cell proliferation, and transformation, as well as tumor development [3, 40–42]. JNK1 and JNK2 possess similarities in structure and biochemistry, and their biological functions that exist in cells overlap. Lacking JNK1 reduces the susceptibility to DEN-induced liver tumor development [42]. Deletion of IKK in hepatocytes increases DEN-induced hepatocyte death and cytokine-driven compensatory proliferation, and disruption of JNK1 abrogates this response [41, 42]. Hepatocyte compensatory proliferation requires more protein synthesis, which needs to elevate the expression of Brf1 and Pol III genes to meet the requirement of cell growth and proliferation. It shows that JNKs may modulate Brf1 expression and Pol III gene transcription in these cellular processes.As mentioned above, JNK1 and JNK2 have overlapping functions in cells. However, their roles in cell proliferation and transformation also appear different [3]. Jnk2-/- fibroblasts display earlier entering into the S phase of the cell cycle, but the performance of jnk1-/- fibroblasts appear to be the inverse case [43]. JNK1 deficiency attenuates c-Jun phosphorylation and causes instability of fibroblasts while restoring the JNK1 expression construct into the cells reverses the JNK1 null phenotype [42]. In contrast, knockout of JNK2 enhances c-Jun phosphorylation and its stability, and reexpressing JNK2 in the jnk2-/- cells plays the opposite role [42]. Further studies have indicated that JNK1 and JNK2 differently modulate TFIIIB subunit (TBP, Brf1, and Bdp1) expression and Pol III gene transcription [44, 45]. Blocking JNK1 expression in jnk1-/- MEFs reduces the cellular levels of TBP, Brf1, and Bdp1, but JNK2 deficiency enhances the levels of TFIIIB subunits in the cells [44]. In contrast, increasing JNK1 expression by its construct augments the cellular level of TBP and Pol III gene transcription, while enhancement of JNK2 expression in its null cells displays lower levels of TBP expression and Pol III gene transcription [44, 45]. The level alterations of TFIIIB subunits caused by JNK1 and JNK2 lead to the changes in Pol III gene transcription [45]. These studies have demonstrated that JNK1 positively regulates Pol III gene transcription. In contrast, JNK2 negatively does so [45]. JNK1 and JNK2 differently modulate the expression of TBP, Brf1, and Pol III genes, resulting in the alterations of cell proliferation [44, 45]. It shows that JNK1 and JNK2 differently modulate cell growth through the alterations of TBP, Brf1, and Pol III gene transcription, while repressing cJun expression by its siRNA decreases the cellular levels of TBP and Brf1 [16]. It suggests that cJun plays a direct role in TBP and Brf1 expression.


### 3.3. Alcohol-Activated JNK1 Upregulates Brf1 Expression and Pol III Gene Transcription to Promote Liver Tumor Development

As we know, alcohol metabolism produces ROS to cause cellular stress and tissue injury. Stress is able to activate JNKs. Studies have shown that alcohol induces JNK1 activation higher than JNK2 [16]. In terms of JNK1 positively regulating TFIIIB activity and Pol III gene transcription, it suggests that alcohol-mediated JNK1 activation may modulate TBP, Brf1, and Pol III genes. A signaling study indicates that alcohol markedly induces JNK1 phosphorylation in ADH-HepG2 cells [16, 46]. Inhibiting JNK1 by its chemical inhibitor, SP600125, or its siRNA significantly decreases the activities of TBP, Brf1, and Pol III genes in alcohol-treated ADH-HepG2 cells [16, 46], while enhancing JNK1 expression by its construct increases the activities of these genes [16, 46]. Further analysis reveals that alcohol-activated JNK1 upregulates c-Jun expression and enhances Elk-1 activity, while the latter augments the expression of TBP, Brf1, and Pol III genes [16]. An animal study indicates that alcohol feeding promotes liver tumor development in HCV-NS5A transgenic mice [16], whereas the levels of TBP, Brf1, and Pol III gene expression in liver tumor tissues are dramatically higher than those in nontumor liver tissues of alcohol-fed mice. Repressing JNK1 decreases the rate of alcohol-induced cell transformation [46]. A human subject study further indicates that Brf1 is overexpressed in HCC (hepatocellular carcinoma) and high expression of Brf1 displays a short survival period of the HCC cases [8]. These studies demonstrate that alcohol induces deregulation of Pol III gene transcription through the JNK1-Elk1-cJun-TFIIIB-Pol III gene pathway (Figure 1).Figure 1
Alcohol-induced liver tumor development. Alcohol induces JNK1 activation to upregulate c-Jun expression through Elk-1. Elk-1 and c-Jun augment TBP and Brf1 expression to enhance Pol III gene transcription, resulting in liver tumor development. Blue P: phosphorylation group.


### 3.4. JNK1-Mediated ERα and Runx2 Expression Affects Brf1 and Pol III Gene Transcription to Cause Phenotypic Changes in Breast Cancer Cells

Signaling analysis indicates that alcohol induces activation of JNK1 in ER+ breast cancer cells [4]. Inhibition of JNK1 by its chemical inhibitor, SP600125, or its siRNA decreases the promoter activity of ERα and also reduces the cellular levels of ERα mRNA and protein [4]. In contrast, enhancing JNK1 expression or activated JNK1 increases the cellular level of ERα [4]. JNK1-caused ERα alteration results in the corresponding changes in Brf1 and Pol III gene transcription [4]. Alcohol induces these changes in ERα, Brf1, and Pol III genes, leading to the alteration of cell phenotypes [4, 5].More interestingly, alcohol also induces a similar alteration of Runx2 in the cells through the JNK1 pathway [7]. Repressing ERα activity weakens alcohol-caused elevations of Runx2 expression [7]. Repressing the expression of ERα, Runx2, or Brf1 attenuates the rates of alcohol-induced colony formation. Together, the signaling pathway of alcohol-induced deregulation of Brf1 and Pol III genes in ER+ breast cancer cells is that alcohol activates JNK1 to increase the levels of ERα and Runx2, resulting in the upregulation of Brf1 and Pol III gene transcription, eventually causing cell proliferation and transformation and breast cancer development (Figure 2) [4, 5, 7, 46].Figure 2
Alcohol-activated JNK1 mediates ERα and Runx2. Alcohol induces JNK1 activation to increase the expression of ERα and Runx2, resulting in deregulation of Pol III genes to promote breast cancer development. Blue P: phosphorylation group.


## 3.1. MAPKs Mediate RNA Pol I- and Pol III-Dependent Gene Transcription through TBP

EGF is a ligand of EGFR (epidermal growth factor receptor). EGF increases TBP (TATA-binding protein) expression [34]. The latter is a general transcription factor, which participates in the regulation of RNA Pol I, Pol II, and Pol III gene transcription. EGF enhances TBP expression to upregulate RNA Pol I and Pol III gene transcription [34]. Mutated Ras decreases EGF-induced TBP promoter activity while blocking the EGFR pathway inhibits TBP expression [34, 35]. Repressing TBP expression results in a decrease in EGF-induced Pol I and Pol III gene transcription [34]. EGF induces phosphorylation of MAP kinases (ERKS, p38 kinases, and JNKs), whereas their chemical inhibitors (U0126, SB202910, and SP600125) of ERKs, p38 kinases, and JNKs inhibit EGF-induced TBP promoter activity, respectively [34]. In addition, expressing dominant-negative mutant forms of ERK2, p38, or JNK1 also block TBP promoter activity [34]. The further studies by different long fragments of the TBP promoter show that EGF-increased activity of the TBP promoter region targets the site of Ets through the MAP kinase pathway [34]. Once Ets is mutated, EGF lost the role in enhancement in the TBP promoter activity [34]. These studies clearly indicate that all of the three subfamilies of MAPKs are involved in EGF-induced TBP expression and RNA Pol I and RNA Pol III gene transcription. EGF-induced transcriptions of these genes are through the EGFR-Ras-MAPK pathway.


## 3.2. JNK1 and JNK2 Differently Modulate Brf1 Expression and Pol III Gene Transcription

JNKs are a subfamily of MAPKs, which include the three members of this subfamily: JNK1, JNK2, and JNK3, which are encoded byjnk1, jnk2, and jnk3 genes, respectively, to produce multiple isoforms through different splicing [36–38]. JNK1 and JNK2 are universally expressed in all organs, but JNK3 is expressed in a few tissues, such as the heart, testis, and brain [37]. JNKs are activated by stress, inflammation, heat shock, carcinogens, and mitogens [38, 39]. Stimulators activate JNKs, namely, phosphorylated JNKs which participate in complex cellular processes, such as inflammation, cell mitogenesis, apoptosis, cell proliferation, and transformation, as well as tumor development [3, 40–42]. JNK1 and JNK2 possess similarities in structure and biochemistry, and their biological functions that exist in cells overlap. Lacking JNK1 reduces the susceptibility to DEN-induced liver tumor development [42]. Deletion of IKK in hepatocytes increases DEN-induced hepatocyte death and cytokine-driven compensatory proliferation, and disruption of JNK1 abrogates this response [41, 42]. Hepatocyte compensatory proliferation requires more protein synthesis, which needs to elevate the expression of Brf1 and Pol III genes to meet the requirement of cell growth and proliferation. It shows that JNKs may modulate Brf1 expression and Pol III gene transcription in these cellular processes.As mentioned above, JNK1 and JNK2 have overlapping functions in cells. However, their roles in cell proliferation and transformation also appear different [3]. Jnk2-/- fibroblasts display earlier entering into the S phase of the cell cycle, but the performance of jnk1-/- fibroblasts appear to be the inverse case [43]. JNK1 deficiency attenuates c-Jun phosphorylation and causes instability of fibroblasts while restoring the JNK1 expression construct into the cells reverses the JNK1 null phenotype [42]. In contrast, knockout of JNK2 enhances c-Jun phosphorylation and its stability, and reexpressing JNK2 in the jnk2-/- cells plays the opposite role [42]. Further studies have indicated that JNK1 and JNK2 differently modulate TFIIIB subunit (TBP, Brf1, and Bdp1) expression and Pol III gene transcription [44, 45]. Blocking JNK1 expression in jnk1-/- MEFs reduces the cellular levels of TBP, Brf1, and Bdp1, but JNK2 deficiency enhances the levels of TFIIIB subunits in the cells [44]. In contrast, increasing JNK1 expression by its construct augments the cellular level of TBP and Pol III gene transcription, while enhancement of JNK2 expression in its null cells displays lower levels of TBP expression and Pol III gene transcription [44, 45]. The level alterations of TFIIIB subunits caused by JNK1 and JNK2 lead to the changes in Pol III gene transcription [45]. These studies have demonstrated that JNK1 positively regulates Pol III gene transcription. In contrast, JNK2 negatively does so [45]. JNK1 and JNK2 differently modulate the expression of TBP, Brf1, and Pol III genes, resulting in the alterations of cell proliferation [44, 45]. It shows that JNK1 and JNK2 differently modulate cell growth through the alterations of TBP, Brf1, and Pol III gene transcription, while repressing cJun expression by its siRNA decreases the cellular levels of TBP and Brf1 [16]. It suggests that cJun plays a direct role in TBP and Brf1 expression.


## 3.3. Alcohol-Activated JNK1 Upregulates Brf1 Expression and Pol III Gene Transcription to Promote Liver Tumor Development

As we know, alcohol metabolism produces ROS to cause cellular stress and tissue injury. Stress is able to activate JNKs. Studies have shown that alcohol induces JNK1 activation higher than JNK2 [16]. In terms of JNK1 positively regulating TFIIIB activity and Pol III gene transcription, it suggests that alcohol-mediated JNK1 activation may modulate TBP, Brf1, and Pol III genes. A signaling study indicates that alcohol markedly induces JNK1 phosphorylation in ADH-HepG2 cells [16, 46]. Inhibiting JNK1 by its chemical inhibitor, SP600125, or its siRNA significantly decreases the activities of TBP, Brf1, and Pol III genes in alcohol-treated ADH-HepG2 cells [16, 46], while enhancing JNK1 expression by its construct increases the activities of these genes [16, 46]. Further analysis reveals that alcohol-activated JNK1 upregulates c-Jun expression and enhances Elk-1 activity, while the latter augments the expression of TBP, Brf1, and Pol III genes [16]. An animal study indicates that alcohol feeding promotes liver tumor development in HCV-NS5A transgenic mice [16], whereas the levels of TBP, Brf1, and Pol III gene expression in liver tumor tissues are dramatically higher than those in nontumor liver tissues of alcohol-fed mice. Repressing JNK1 decreases the rate of alcohol-induced cell transformation [46]. A human subject study further indicates that Brf1 is overexpressed in HCC (hepatocellular carcinoma) and high expression of Brf1 displays a short survival period of the HCC cases [8]. These studies demonstrate that alcohol induces deregulation of Pol III gene transcription through the JNK1-Elk1-cJun-TFIIIB-Pol III gene pathway (Figure 1).Figure 1
Alcohol-induced liver tumor development. Alcohol induces JNK1 activation to upregulate c-Jun expression through Elk-1. Elk-1 and c-Jun augment TBP and Brf1 expression to enhance Pol III gene transcription, resulting in liver tumor development. Blue P: phosphorylation group.


## 3.4. JNK1-Mediated ERα and Runx2 Expression Affects Brf1 and Pol III Gene Transcription to Cause Phenotypic Changes in Breast Cancer Cells

Signaling analysis indicates that alcohol induces activation of JNK1 in ER+ breast cancer cells [4]. Inhibition of JNK1 by its chemical inhibitor, SP600125, or its siRNA decreases the promoter activity of ERα and also reduces the cellular levels of ERα mRNA and protein [4]. In contrast, enhancing JNK1 expression or activated JNK1 increases the cellular level of ERα [4]. JNK1-caused ERα alteration results in the corresponding changes in Brf1 and Pol III gene transcription [4]. Alcohol induces these changes in ERα, Brf1, and Pol III genes, leading to the alteration of cell phenotypes [4, 5].More interestingly, alcohol also induces a similar alteration of Runx2 in the cells through the JNK1 pathway [7]. Repressing ERα activity weakens alcohol-caused elevations of Runx2 expression [7]. Repressing the expression of ERα, Runx2, or Brf1 attenuates the rates of alcohol-induced colony formation. Together, the signaling pathway of alcohol-induced deregulation of Brf1 and Pol III genes in ER+ breast cancer cells is that alcohol activates JNK1 to increase the levels of ERα and Runx2, resulting in the upregulation of Brf1 and Pol III gene transcription, eventually causing cell proliferation and transformation and breast cancer development (Figure 2) [4, 5, 7, 46].Figure 2
Alcohol-activated JNK1 mediates ERα and Runx2. Alcohol induces JNK1 activation to increase the expression of ERα and Runx2, resulting in deregulation of Pol III genes to promote breast cancer development. Blue P: phosphorylation group.


## 4. The Role of MSK1 in Alcohol-Induced Deregulation of Brf1 and Pol III Gene Transcription

As discussed above, MAPKs (ERKs, p38 kinases, and JNKs) play vital roles in EGF-induced TFIIIB activity and RNA Pol I and Pol III gene transcription, which is through the EGFR-Ras pathway. Further analysis shows that alcohol induces the deregulation of Brf1 expression and Pol III gene transcription to cause the alteration of cell phenotypes in liver and breast cells which is mainly through the MAPK subfamily, JNK1 pathway [4, 16, 46]. Therefore, we further discuss the downstream of MAPKs and how it mediates alcohol-induced responses.MSK1 (mitogen- and stress-activated protein kinase 1) is a protein kinase of serine/threonine residuals in nuclei. MSK1 is also called nuclear kinase which plays critical roles in chromatin remodeling and gene transcription through MSK1-mediated phosphorylation of histone H3 under stress conditions [47–49]. MSK1 is a downstream component of the MAPK pathway. Studies have demonstrated that MSK1 regulates gene expression and cell transformation [49–53]. An MSK knockout mouse has no significant health problems, while MSK deficiency inhibits skin cancer development of mice [54, 55]. Earlier studies have demonstrated that phosphorylated histone H3 mediates Brf1 expression and Pol III gene transcription [14, 56]. It suggests that MSK1 may participate in the regulation of Brf1 and Pol III gene activities. A recent study indicates that alcohol induces phosphorylation of MSK1 at serine 376 and threonine 581 to activate it [10]. Inhibiting MSK1 attenuates alcohol-induced Brf1 promoter activity in HepG2-ADH cells. This suggests that MSK1 may take part in the regulation of alcohol-induced Brf1 transcription [10]. The truncated-fragment analysis of the Brf1 promoter reporter reveals that the high peak of MSK1-mediated Brf1 promoter activity is at the site of p-328/+109 bp [10]. More interestingly, blocking MSK1 reduces alcohol-caused elevation of Brf1 expression and Pol III gene transcription, resulting in the attenuation of the rates of proliferation and colony formation of HepG2-ADH cells treated with alcohol [10]. These studies have demonstrated that MSK1 mediates alcohol-induced deregulation of Brf1 and Pol III genes.


## 5. Deregulation of Brf1 and Pol III Genes Is Mediated by pAMPKα in Lung Cancer

AMPK (5′ AMP-activated protein kinase or 5′ adenosine monophosphate-activated protein kinase) is a key downstream component of a tumor suppressor, LKB1, while mutations of LKB1 are found in over 20% of patients with NSCLC (non-small-cell lung cancer) and frequently associated with activating K-RAS mutations [57–59]. AMPK is composed of three subunits (α, β, and γ) to form a heterotrimeric protein complex. The phosphorylation of these subunits plays a vital role in AMPK activity and its stability [60]. AMPK enhances glucose and fatty uptake and β-oxidation [61]. AMPK also decreases the synthesis of cholesterol, triglycerides, and fatty acids [61]. It shows that AMPK participates in the regulation of energy metabolism. ROS-induced oxidative stress activates the JNK1 pathway to increase Brf1 expression [16]. A recent study also indicates that levels of ROS of lung cancer cells are associated with the alteration of pAMPKα (phosphorylated AMPKα) [62], while AMPK activation is associated with protein synthesis [60, 61]. The process of protein synthesis is controlled by Brf1 and Pol III genes. As LKB1 is often mutated in the cases of human lung cancer, this implies that ROS and AMPK are potentially involved in the process of Brf1 expression and Pol III gene transcription, which may be related to lung cancer development.A very recent study indicates that Brf1 expression is increased in most cases of human lung cancer [63]. The Brf1 overexpression is accompanied by a high level of pAMPKα in the cases of lung cancer [63]. Mechanism analysis reveals that carcinogen MNNG, an agent of DNA damage, induces pAMPKα in lung cancer cells. Brf1 expression and Pol III gene transcription are increased in MNNG-treated lung cancer cells. Inhibiting pAMPKα signaling attenuates MNNG-increased expression of Brf1 and Pol III genes while repressing AMPKα and Brf1 by their siRNAs decreases the rates of MNNG-promoted cell proliferation and colony formation [63]. These studies demonstrate that pAMPKα takes part in the modulation of Brf1 expression and Pol III gene transcription. pAMPKα plays a critical role in the transcription of these genes and lung cancer development.


## 6. PLK1 Phosphorylates Brf1 to Affect Pol III Gene Transcription

Except for the signaling alteration of Brf1 expression induced by carcinogens or dietary factors mentioned above, Brf1 modification plays a vital role in Pol III gene transcription too. PLK1 (polo-like kinase 1, also called serine/threonine-protein kinases) is located in the centrosome in the interphase and is associated with mitotic spindle poles [64]. PLK1 plays important roles in the cell cycle, chromosome separation, and centrosome forming [65]. PLK1 is also known as a protooncogene, and it is often overexpressed in tumor cells. Overexpression of PLK1 promotes tumor formation in nude mice [66], while the tumor suppressor pRB is able to repress PLK1 activity. Studies have indicated that PLK1 is associated with colon, lung cancer and leukemia [67–69].Phosphorylation of TFIIIB (TBP, Brf1, and Bdp1) is often associated with the changes in Pol III gene transcription. Inactivation of TFIIIB subunits during mitosis is through their phosphorylation [70–72]. Studies have demonstrated that PLK1 directly phosphorylates Brf1 at serine 450 to enhance Pol III gene transcription at the interphase of the cell cycle, which is consistent with its stimulating role in cell proliferation [73]. In contrast, inhibiting PLK1 activity by its inhibitor BI2536 reduces the transcription [73]. Increasing PLK1 activity enhances phosphorylation of Brf1 at threonine 270 to prevent RNA Pol III recruitment at Pol III genes during mitosis [73]. These studies indicate that phosphorylation of Brf1 at the two sites (S450 and T270) reveals distinct effects on Pol III gene transcription at different phases of the cell cycle.


## 7. Other Signaling Events Which Mediate Activities of Brf1 and Pol III Genes

BRCA1 (breast cancer susceptibility gene 1) is a tumor suppressor [74]. Normally, BRCA1 repairs DNA damage to repress tumor development in breast tissue. Once BRCA1 is mutated, it loses this function, while the damaged DNA cannot be properly repaired. This enhances the risk of breast cancer in women [75]. Moreover, studies have demonstrated that transfecting wild-type BRCA1 expression constructs into the BRCA1-deficient cells decreases Pol III gene transcription [17], while expressing truncated or mutated BRCA1 does not affect this transcription in the cells [17]. Further study reveals that alcohol does not change the cellular level of BRCA1, but overexpressing BRCA1 attenuates alcohol-induced Pol III gene transcription in ER+ breast cancer cells [17]. The samples of gastric cancer patients with alcohol intake indicate that overexpression of Brf1 is in all of these samples, while the BRCA1 expression rate is 62.3% (48 of 77) [11]. Both Brf1 and BRCA1 colocalize in nuclei [11]. Interestingly, the data reveals that the expression of Brf1 and BRCA1 is closely associated with the enhancement of alcohol consumption [11]. The high Brf1 expression displays shorter disease-free survival times than low levels of Brf1 in the cases of gastric cancer. It suggests that patients of gastric cancer with alcohol consumption reveal a worse prognosis [11]. As mentioned above, alcohol does not affect BRCA1 expression in the cell lines of breast cancer [17], while high levels of BRCA1 are determined in the samples of gastric patients with alcohol consumption [11]. It seems a contradictory phenomenon. This phenomenon may be associated with the two various systems of in vitro and in vivo. In the cells, alcohol-induced signaling does not influence the cellular level of BRCA1, while in patient’ samples, there exists mutation of BRCA1 which loses the function repressing Brf1 and Pol III genes. More details need to be further investigated.PTEN (phosphatase and tensin homolog) is another tumor suppressor. Study shows that PI3K (phosphatidylinositol 3-kinase)/PETN is also involved in the regulation of Brf1 and Pol III gene transcription. Restoring PTEN into PTEN-deficient cells inhibits Pol III gene transcription [76]. In contrast, decreasing PTEN expression augments the transcription of Pol III genes [76]. The analysis of transcription machinery indicates that PTEN inhibits serine phosphorylation of Brf1 to reduce the occupancy of TFIIIB complexes of tRNA genes. PTEN targets TFIIIB complexes to disrupt the association of TBP and Brf1 to repress Pol III gene transcription. In addition, prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1 [76]. This process is through inactivation of the PI3K/PTEN/mTOR pathway [76].


## 8. Summary

The stimulators, carcinogens (EGF, DEN, and MNNG) and dietary factors (alcohol), induce ROS release in cells. ROS causes cellular phenotypic alterations, such as cell death, stress, apoptosis, cell proliferation, transformation, and eventually tumor development through different signaling pathways which mediate the deregulation of Brf1 and Pol III gene transcription (Figure3), whereas the deregulation of these genes is tightly linked to the alteration of these cellular phenotypes. In this paper, we summarize our works in the past dozen years and introduce the changes in Brf1 expression and Pol III gene transcription, which are mediated by various signaling pathways. These studies indicate that Brf1 and Pol III genes are novel biological targets of ROS. Deregulation of these genes is tightly linked to cell transformation and human cancers, while the risk of human cancer is steeply increased with enhancing alcohol intake daily [12]. Therefore, reduction of alcohol consumption is to mitigate the risk of human cancers [12]. On the other hand, developing a specific inhibitor to repress the activities of Brf1 and Pol III genes may be an efficient approach to attenuate the incidence of human cancers, particularly in people who consume alcohol.Figure 3
Schematic illustration of ROS-mediated deregulation of Brf1 and Pol III gene transcription. ROS causes deregulation of Brf1 expression and RNA Pol III gene transcription through various pathways, resulting in the alterations of cellular phenotypes, eventually cancer development.


## 9. Clinical Significance of Signaling Events Mediating Brf1 and Pol III Gene Transcription

As mentioned above, upregulation of Brf1 and Pol III genes is tightly linked to cell transformation and human cancer [5–8], once inhibition of Brf1 and Pol III gene transcription represses cell transformation of nontumor cells and colony formation of tumor cells by blocking the pathways which involve the activities of Brf1 and Pol III genes [4–8, 63]. These studies strongly suggest a possibility to develop a specific inhibitor for a cancer therapy or mixed inhibitors for multiple human cancers. Therefore, developing new inhibitor(s) by reducing the levels of Brf1 expression and Pol III gene transcription shows a potential approach for human cancer therapy. We are working in this area and obtained some interesting progress in a tissue culture model and animal model (unpublished). We expect that more progress will be gained during building the new approaches. It will benefit the patients who are enduring the pain of cancers.



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*Source: 5888432-2021-10-04.xml*