Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure.

Since invasive bladder cancer (BC) can progress to life threatening metastases, understanding the molecular mechanisms underlying BC invasion is crucial for potentially decreasing the mortality of this disease. Herein, it is discovered that autophagy‐related gene 7 (ATG7) is remarkably overexpressed in human invasive BC tissues. The knockdown of ATG7 in human BC cells dramatically inhibits cancer cell invasion, revealing that ATG7 is a key player in regulating BC invasion. Mechanistic studies indicate that MIR190A is responsible for ATG7 mRNA stability and protein overexpression by directly binding to ATG7 mRNA 3′‐UTR. Furthermore, ATG7‐mediated autophagy promotes HNRNPD (ARE/poly(U)‐binding/degradation factor 1) protein degradation, and in turn reduces HNRNPD interaction with ARHGDIB mRNA, resulting in the elevation of ARHGDIB mRNA stability, and subsequently leading to BC cell invasion. The identification of the MIR190A/ATG7 autophagic mechanism regulation of HNRNPD/ARHGDIB expression provides an important insight into understanding the nature of BC invasion and suggests that autophagy may represent a potential therapeutic strategy for the treatment of human BC patients.

Background: MicroRNAs (miRNAs), the key regulator of gene expression, and N6‐methyladenosine (m6A) RNA modification play a significant role in tumour progression. However, regulation of m6A‐modified mRNAs by miRNAs in colorectal cancer (CRC), and its effect on progression of CRC, remains to be investigated.Methods: Expression of miR‐6125 and YTH Domain‐Containing Family Protein 2 (YTHDF2) was detected by western blotting and immunohistochemistry. The effects of miR‐6125 and YTHDF2 on proliferative capacity of CRC cells were analysed using soft agar, ATP, CCK8 and EdU assays, and in animal experiments.Results: MiR‐6125 expression was downregulated markedly in CRC, and expression correlated negatively with tumour size and prognosis. MiR‐6125 targeted the 3′‐UTR of YTHDF2 and downregulated the YTHDF2 protein, thereby increasing the stability of m6A‐modified glycogen synthase kinase 3 beta (GSK3β) mRNA. Increased GSK3β protein levels inhibited the expression of Wnt/β‐catenin/Cyclin D1 pathway‐related proteins, leading to G0‐G1 phase arrest and ultimately inhibiting the proliferation of CRC cells.Conclusions: MiR‐6125 regulates YTHDF2 and thus plays a critical role in regulating the Wnt/β‐catenin pathway, thereby affecting the growth of CRC. Collectively, these results suggest that miR‐6125 and YTHDF2 are potential targets for treatment of CRC.

Purpose: The carcinogenic capacity of B[a]P/B[a]PDE is supported by epidemiologic studies. However, the molecular mechanisms responsible for B[a]P/B[a]PDE-caused lung cancer have not been well investigated. We evaluated here the role of novel target PHLPP2 in lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. Experimental Design: We used the Western blotting, RT-PCR, [³⁵S]methionine pulse and immunohistochemistry staining to determine PHLPP2 downregulation following B[a]P/B[a]PDE exposure. Both B[a]PDE-induced Beas-2B cell transformation model and B[a]P-caused mouse lung cancer model were used to elucidate the mechanisms leading to PHLPP2 downregulation and lung carcinogenesis. The important findings were also extended to in vivo human studies. Results: We found that B[a]P/B[a]PDE exposure downregulated PHLPP2 expression in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The ectopic expression of PHLPP2 dramatically inhibited cell transformation upon B[a]PDE exposure. Mechanistic studies showed that miR-205 induction was crucial for inhibition of PHLPP2 protein translation by targeting PHLPP2-3′-UTR. Interestingly, PHLPP2 expression was inversely associated with tumor necrosis factor alpha (TNFα) expression, with low PHLPP2 and high TNFα expression in lung cancer tissues compared with the paired adjacent normal lung tissues. Additional studies revealed that PHLPP2 exhibited its antitumorigenic effect of B[a]P/B[a]PDE through the repression of inflammatory TNFα transcription. Conclusions: Our studies not only first time identify PHLPP2 downregulation by lung carcinogen B[a]P/B[a]PDE, but also elucidate a novel molecular mechanisms underlying lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. Clin Cancer Res; 21(16); 3783–93. ©2015 AACR.

Since invasive bladder cancer (BC) is one of the most lethal urological malignant tumors worldwide, understanding the molecular mechanisms that trigger the migration, invasion, and metastasis of BC has great significance in reducing the mortality of this disease. Although RelA/p65, a member of the NF-kappa B transcription factor family, has been reported to be upregulated in human BCs, its regulation of BC motility and mechanisms have not been explored yet. NF-κBp65 expression was evaluated in N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced high invasive BCs by immunohistochemistry staining and in human BC cell lines demonstrated by Western Blot. The effects of NF-κBp65 knockdown on BC cell migration and invasion, as well as its regulated RhoGDIα and FBW7, were also evaluated in T24T cells by using loss- and gain-function approaches. Moreover, the interaction of FBW7 with RhoGDIα was determined with immunoprecipitation assay, while critical role of ubiquitination of RhoGDIα by FBW7 was also demonstrated in the studies. p65 protein was remarkably upregulated in the BBN-induced high invasive BCs and in human BC cell lines. We also observed that p65 overexpression promoted BC cell migration by inhibiting RhoGDIα expression. The regulatory effect of p65 on RhoGDIα expression is mediated by its upregulation of FBW7, which specifically interacted with RhoGDIα and promoted RhoGDIα ubiquitination and degradation. Mechanistic studies revealed that p65 stabilizing the E3 ligase FBW7 protein was mediated by its attenuating pten mRNA transcription. We demonstrate that p65 overexpression inhibits pten mRNA transcription, which stabilizes the protein expression of ubiquitin E3 ligase FBW7, in turn increasing the ubiquitination and degradation of RhoGDIα protein and finally promoting human BC migration. The novel identification of p65/PTEN/FBW7/RhoGDIα axis provides a significant insight into understanding the nature of BC migration, further offering a new theoretical support for cancer therapy.

NF-κB is a well-known transcription factor in regulation of multiple gene transcription and biological processes, and most of them are relied on its transcriptional activity of the p65/RelA subunit, while biological function of another ubiquitously expressed subunit NF-κB1 (p50) remains largely unknown due to lack transcriptional activation domain. Here we discovered a novel biological function of p50 as a regulator of oncogenic c-Myc protein degradation upon arsenite treatment in a NF-κB transcriptional-independent mechanism. Our results found that p50 was crucial for c-Myc protein induction following arsenite treatment by using specific knockdown and deletion of p50 in its normal expressed cells as well as reconstituting expression of p50 in its deficient cells. Subsequently we showed that p50 upregulated c-Myc protein expression mainly through inhibiting its degradation. We also identified that p50 exhibited this novel property by suppression of FBW7 expression. FBW7 was profoundly upregulated in p50-defecient cells in comparison to that in p50 intact cells, whereas knockdown of FBW7 in p50-/- cells restored arsenite-induced c-Myc protein accumulation, assuring that FBW7 up-regulation was responsible for defect of c-Myc protein expression in p50-/- cells. In addition, we discovered that p50 suppressed fbw7 gene transcription via inhibiting transcription factor E2F1 transactivation. Collectively, our studies demonstrated a novel function of p50 as a regulator of c-Myc protein degradation, contributing to our notion that p50-regulated protein expression through multiple levels at protein translation and degradation, further providing a significant insight into the understanding of biomedical significance of p50 protein.