Smoking-induced control of miR-133a-3p alters the expression of EGFR and HuR in HPV-infected oropharyngeal cancer.

PURPOSE: Human papillomavirus (HPV) infected oropharyngeal squamous cell carcinoma (OPSCC) patients have a better prognosis compared to HPV(-) counterparts. However, a subset of HPV(+) patients with a smoking history fail to respond to the standard of care treatments such as radiation and chemotherapy. To understand the underlying mechanism driving HPV(+) OPSCC patient resistance to treatment and recurrence, we sought to identify and characterize the differentially expressed miRNAs and their target genes in HPV(+) smokers and non-smokers. EXPERIMENTAL DESIGN: MicroRNA expression analysis was performed using Nanostring in tumor tissues isolated from a prospective cohort of HPV(+) smoking (n = 9) and HPV(+) (n = 13) non-smoking OPSCC patients. Identified miRNAs of interest were further validated using qRT-PCR in cigarette smoke extract (CSE) treated HPV(+) and E6/E7 overexpressing HPV(-) cells. RESULTS: In comparison to OPSCC HPV(+) non-smokers, 38 miRNAs were significantly altered in the HPV(+) smoker patients cohort and out of that 9 were downregulated. Altered miRNA expression was also detected in the serum and metastatic lymph nodes of HPV(+) smokers versus non-smokers. Expression of miR-133a-3p was significantly downregulated in OPSCC smokers, HPV(+) cells and E6/E7 overexpressing HPV(-) cells treated with CSE. Reduction of miR-133a-3p induced the upregulation of miR-133a-3p target mRNAs EGFR and HuR. CONCLUSIONS: Our results indicate that miR-133a-3p is a target of smoking-induced changes in HPV(+) patients and alters the expression of EGFR and HuR which may promote HPV associated oropharyngeal cancer. Therefore, future treatment strategies for HPV(+) OPSCC smokers should focus on EGFR inhibition and the development of selective therapies to target HuR.

The Long (lncRNA) and Short (miRNA) of It: TGFß-Mediated Control of RNA-Binding Proteins and Noncoding RNAs.

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFß) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFß signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFß signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer Res; 16(4); 567-79. ©2018 AACR.

Correction: RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

[This corrects the article DOI: 10.1371/journal.pgen.1006306.].

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.

RNA-binding protein CELF1 promotes tumor growth and alters gene expression in oral squamous cell carcinoma.

The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention.

Inhibition of caspases protects mice from radiation-induced oral mucositis and abolishes the cleavage of RNA-binding protein HuR.

The oral mucosal epithelium is typically insulted during chemotherapy and ionizing radiation (IR) therapy and disposed to mucositis, which creates painful inflammation and ulceration in the oral cavity. Oral mucositis alters gene expression patterns, inhibits cellular growth, and initiates cell death in the oral epithelial compartments. Such alterations are governed by several different factors, including transcription factors, RNA-binding proteins, and microRNAs. IR-induced post-transcriptional regulation of RNA-binding proteins exists but is poorly studied in clinically relevant settings. We herein report that the RNA-binding protein human antigen R (HuR) undergoes cleavage modification by caspase-3 following IR-induced oral mucositis and subsequently promotes the expression of the pro-apoptotic factor BAX (Bcl-2-associated X protein), as well as cell death. Further analyses revealed that the HuR cleavage product-1 (HuR-CP1) directly associates and stabilizes the BAX mRNA and concurrently activates the apoptotic pathway. On the other hand, a noncleavable isoform of HuR promotes the clonogenic capacity of primary oral keratinocytes and decreases the effect of IR-induced cell death. Additionally, specific inhibition of caspase-3 by a compound, NSC321205, increases the clonogenic capacity of primary oral keratinocytes and causes increased basal layer cellularity, thickened mucosa, and elevated epithelial cell growth in the tongues of mice with oral mucositis. This protective effect of NSC321205 is mediated by a decrease in caspase-3 activity and the consequent inhibition of HuR cleavage, which reduces the expression of BAX in mice with IR-induced oral mucositis. Thus, we have identified a new molecular mechanism of HuR in the regulation of mRNA turnover and apoptosis in oral mucositis, and our data suggest that blocking the cleavage of HuR enhances cellular growth in the oral epithelial compartment.

Overexpression of RNA-binding protein CELF1 prevents apoptosis and destabilizes pro-apoptotic mRNAs in oral cancer cells.

CELF1 RNA-binding protein, otherwise called CUGBP1, associates and coordinates the degradation of GU-rich element (GRE) containing mRNA's encoding factors important for cell growth, migration and apoptosis. Although many substrates of CELF1 have been identified, the biological significance of CELF1-mediated mRNA decay remains unclear. As the processes modulated by CELF1 are frequently disrupted in cancer, we investigated the expression and role of CELF1 in oral squamous cancer cells (OSCCs). We determined that CELF1 is reproducibly overexpressed in OSCC tissues and cell lines. Moreover, depletion of CELF1 reduced proliferation and increased apoptosis in OSCCs, but had negligible effect in non-transformed cells. We found that CELF1 associates directly with the 3'UTR of mRNAs encoding the pro-apoptotic factors BAD, BAX and JunD and mediates their rapid decay. Specifically, 3'UTR fragment analysis of JunD revealed that the GRE region is critical for binding with CELF1 and expression of JunD in oral cancer cells. In addition, silencing of CELF1 rendered BAD, BAX and JunD mRNAs stable and increased their protein expression in oral cancer cells. Taken together, these results support a critical role for CELF1 in modulating apoptosis and implicate this RNA-binding protein as a cancer marker and potential therapeutic target.

Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion.

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.

S100A4 regulates macrophage chemotaxis.

S100A4, a member of the S100 family of Ca(2+)-binding proteins, is directly involved in tumor metastasis. In addition to its expression in tumor cells, S100A4 is expressed in normal cells and tissues, including fibroblasts and cells of the immune system. To examine the contribution of S100A4 to normal physiology, we established S100A4-deficient mice by gene targeting. Homozygous S100A4(-/-) mice are fertile, grow normally and exhibit no overt abnormalities; however, the loss of S100A4 results in impaired recruitment of macrophages to sites of inflammation in vivo. Consistent with these observations, primary bone marrow macrophages (BMMs) derived from S100A4(-/-) mice display defects in chemotactic motility in vitro. S100A4(-/-) BMMs form unstable protrusions, overassemble myosin-IIA, and exhibit altered colony-stimulating factor-1 receptor signaling. These studies establish S100A4 as a regulator of physiological macrophage motility and demonstrate that S100A4 mediates macrophage recruitment and chemotaxis in vivo.

Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells.

In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

Traffic of genetic information between segmental duplications flanking the typical 22q11.2 deletion in velo-cardio-facial syndrome/DiGeorge syndrome.

Velo-cardio-facial syndrome/DiGeorge syndrome results from unequal crossing-over events between two 240-kb low-copy repeats termed LCR22 (LCR22-2 and LCR22-4) on Chromosome 22q11.2, comprised of modules, each of which are >99% identical in sequence. To delineate regions in the LCR22s that might contain hotspots for 22q11.2 rearrangements, we scanned the interval for increased rates of recombination with the hypothesis that these regions might be more prone to breakage. We generated an algorithm to detect sites of altered recombination by searching for single nucleotide polymorphic positions in BAC clones from different libraries mapped to LCR22-2 and LCR22-4. This method distinguishes single nucleotide polymorphisms from paralogous sequence variants and complex polymorphic positions. Sites of shared polymorphism are considered potential sites of gene conversion or double cross-over between the two LCR22s. We found an inverse correlation between regions of paralogous sequence variants that are unique to a given position within one LCR22 and clusters of shared polymorphic sites, suggesting that these clusters depict altered recombination and not remnants of ancestral single nucleotide polymorphisms. We postulate that most shared polymorphic sites are products of past transfers of DNA information between the LCR22s, suggesting that frequent traffic of genetic material may induce genomic instability in the two LCR22s. We also found that gaps up to 1.5 kb long can be transferred between LCR22s.