Association of cytoplasmic p27 expression with an unfavorable response to cisplatin-based chemotherapy and poor outcomes in non-small cell lung cancer.

Reduced nuclear p27 expression is associated with a poor outcome in various cancers, including non-small cell lung cancer (NSCLC). Cytoplasmic p27 expression was shown to be associated with an unfavorable response to chemotherapy and poor outcomes in some carcinomas, but it has not been well studied in NSCLC. Herein, p27 expression in 219 tumors surgically resected from NSCLC patients was evaluated by immunohistochemistry (IHC). The most common of p27 immunostaining in lung tumors was observed in the cytoplasm (N-/C+, 32 %), followed by negative (N-/C-, 29 %), nucleus (N+/C-, 24 %), and nucleus plus cytoplasm (N+/C+, 15 %). Kaplan-Meier and Cox regression models showed that p27 N-/C+ tumors exhibited the worst overall survival (OS) and relapse-free survival (RFS) among the four categories of tumors. Among 135 of 219 patients who received cisplatin-based chemotherapy, p27 N-/C+ tumors most commonly showed an unfavorable response to cisplatin-based chemotherapy, followed by p27 N-/C- tumors when p27 N+/C- tumors were used as a reference. IHC analysis for phosphorylated extracellular signal-regulated kinase (p-ERK) and Bcl-2 expression in the lung tumors was performed to test whether ERK activation could enhance p27 nuclear export and the expression of Bcl-2 to test whether ERK activation could enhance p27 nuclear export and Bcl-2 expression. The data showed that p-ERK expression was positively correlated with cytoplasmic p27 (N-/C+) and Bcl-2 expression in the lung tumors. Patients with high Bcl-2-expressing tumors treated with cisplatin-based chemotherapy showed unfavorable predictive values in a subset of this study population. Therefore, we suggest that cytoplasmic p27 (N-/C+) via ERK-activated Bcl-2 expression may predict an unfavorable response to cisplatin-based chemotherapy and poor outcomes in NSCLC.

LKB1 loss at transcriptional level promotes tumor malignancy and poor patient outcomes in colorectal cancer.

BACKGROUND: Liver kinase B1 (LKB1) loss by gene mutation, loss of heterozygosity, and promoter methylation rarely occurs in colorectal cancer. We wondered whether LKB1 loss could be deregulated at the transcriptional level to promote tumor progression and poor outcome in colorectal cancer. METHODS: Mechanistic studies were performed in two each of p53 wild-type (HCT116, LoVo) and p53-mutated (SW480, HT29) colon cancer cells to explore whether LKB1 loss could be deregulated by NKX2-1-mediated p53 pathway. LKB1 and NK2 homeobox 1 (NKX2-1) expressions in colorectal tumors were determined by immunohistochemistry, and the prognostic value of both molecules was assessed by Kaplan-Meier test and Cox regression model. RESULTS: Mechanistically, LKB1 loss at the transcriptional level due to alteration of the NKX2-1-mediated p53 pathway promotes invasiveness in colon cancer cells. The cell invasiveness induced by LKB1 loss was nearly suppressed by mammalian target of rapamycin (mTOR) inhibitor (rapamycin and everolimus) and mTOR/AKT dual inhibitor Palomid 529 (P529). Among patients, low LKB1 tumors exhibited shorter overall survival (OS) and relapse-free survival periods than high LKB1 tumors. The highest hazard ratio value for OS and relapse-free survival was observed in wild-type p53 with low LKB1/low NKX2-1 tumors and in mutated p53 with low LKB1/high NKX2-1 tumors when wild-type p53 with high LKB1/high NKX2-1 and mutated p53 with high LKB1/low NKX2-1 tumors were used as references. CONCLUSIONS: LKB1 loss at the transcriptional level via alteration of the NKX2-1/p53 axis promotes cell invasion, consequently resulting in poor outcome in colorectal cancer patients.

Loss of TIMP-3 promotes tumor invasion via elevated IL-6 production and predicts poor survival and relapse in HPV-infected non-small cell lung cancer.

Human papillomavirus (HPV) 16/18 E6 oncoprotein is expressed in lung tumors and is associated with p53 inactivation. The tissue inhibitor of metalloproteinase 3 (TIMP-3) is essential for limiting inflammation; therefore, we expected that TIMP-3 loss might induce chronic inflammation, thereby promoting tumor malignancy as well as poor survival and relapse in patients with HPV-infected non-small cell lung cancer. In this study, the loss of TIMP-3 by loss of heterozygosity and/or promoter hypermethylation was more frequent in HPV16/18 E6-positive tumors than in E6-negative tumors. To explore the possible underlying mechanism, E6-negative TL4 and CL1-0 cells were transfected with an E6 cDNA plasmid. A marked decrease in TIMP-3 expression was caused by promoter hypermethylation via increased DNA (cytosine-5-)-methyltransferase 1 (DNMT1) expression. Mechanistic studies indicated that TIMP-3 loss promoted interleukin-6 (IL-6) production, which led to cell invasion and anchorage-independent growth on soft agar plates. Kaplan-Meier and Cox regression models showed that patients with low-TIMP-3/high-IL-6 tumors had shorter overall survival and relapse-free survival periods when compared with patients with high-TIMP-3/low-IL-6 tumors. In summary, loss of TIMP-3 may increase IL-6 production via the tumor necrosis factor α/nuclear factor κB axis, thereby promoting tumor malignancy and subsequent relapse and poor survival in patients with HPV-infected non-small cell lung cancer.

Cytoplasmic Ape1 expression elevated by p53 aberration may predict survival and relapse in resected non-small cell lung cancer.

BACKGROUND: Subcellular localization of apurinic/apyrimidinic endonuclease-1/redox factor-1 (Ape1) has been demonstrated to promote lung tumor malignancy via NF-κB activation. We hypothesized that increased cytoplasmic Ape1 expression might cause NF-κB activation by p53 aberration, and result in poor outcome in non-small cell lung cancer (NSCLC). METHODS: Herein, knockdown of E6 or p53 and overexpression of E6 were performed in various lung cancer cells to test whether cytoplasmic Ape1 expression could be elevated by p53 aberration. To examine whether cytoplasmic Ape1 could be associated with patients' outcome, 125 lung tumors from patients with NSCLC were collected to determine Ape1 protein and mRNA expression by immunohistochemistry and real-time RT-PCR. RESULTS: Our data showed that cytoplasmic Ape1 decreased in E6-knockdown TL-1 cells and increased in E6-overexpressed TL-4 and p53-knockdown H520 cells; and cell invasion capability was dependent on the presence of cytoplasmic Ape1. Increases in cytoplasmic Ape1 by p53 aberration may be through activation of Ape1 transcription and S-nitrosation of Ape1 protein. Kaplan-Meier and Cox models showed that patients with high cytoplasmic Ape1 had shorter cancer-specific survival (CSS) and relapse-free survival (RFS) periods than did those with low cytoplasmic Ape1. CONCLUSIONS: We suggest that cytoplasmic Ape1 expression elevated by p53 aberration may be used to predict poor survival and relapse in patients with NSCLC.

Rapid Improvement in Neck Disability, Mobility, and Sleep Quality with Chronic Neck Pain Treated by Fu's Subcutaneous Needling: A Randomized Control Study.

Background: Chronic neck pain is a common musculoskeletal disorder caused by overuse of neck and upper back muscles or poor posture, and it is commonly combined with a limited range of motion in the neck and shoulders. Most cases will recover within a few days; however, the symptoms often recur easily. Fu's subcutaneous needling (FSN) is a new therapeutic approach used to treat patients with chronic neck pain. However, there is no solid evidence to support the effectiveness of FSN on chronic neck pain and disability. Methods: Participants (n = 60) with chronic neck pain for more than 2 months with pain intensity scored by visual analog scale (VAS) more than five were enrolled in this trial. Participants were equally randomized into the FSN or transcutaneous electrical nerve stimulation (TENS) group who received interventions once a day on day 1, day 2, and day 4. They were assessed by outcome measurements during pre- and post-treatment and followed up for 15 days. Results: The VAS was immediately reduced in the FSN and TENS groups and sustained for 15 days of follow-up (all P < 0.001). The immediate effects were also observed as the pressure pain threshold increased in the FSN group on day 2 (P=0.006) and day 4 (P=0.023) after treatment, and tissue hardness decreased by FSN on day 1 and day 2 after treatment (both P < 0.001). FSN and TENS treatment improved neck disability and mobility; moreover, FSN promoted participants to receive better sleep quality, as determined by PSQI assessment (P=0.030). TENS had no benefit on sleep quality. Conclusion: FSN was able to relieve pain and relax muscle tightness. Notably, FSN significantly improved neck disability and mobility and enhanced sleep quality. These findings demonstrated that FSN could be an effective alternative treatment option for patients with chronic neck pain. Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT03605576, registered on July 30, 2018.

Characterization of initial key steps of IL-17 receptor B oncogenic signaling for targeted therapy of pancreatic cancer.

The members of the interleukin-17 (IL-17) cytokine family and their receptors were identified decades ago. Unlike IL-17 receptor A (IL-17RA), which heterodimerizes with IL-17RB, IL-17RC, and IL-17RD and mediates proinflammatory gene expression, IL-17RB plays a distinct role in promoting tumor growth and metastasis upon stimulation with IL-17B. However, the molecular basis by which IL-17RB promotes oncogenesis is unknown. Here, we report that IL-17RB forms a homodimer and recruits mixed-lineage kinase 4 (MLK4), a dual kinase, to phosphorylate it at tyrosine-447 upon treatment with IL-17B in vitro. Higher amounts of phosphorylated IL-17RB in tumor specimens obtained from patients with pancreatic cancer correlated with worse prognosis. Phosphorylated IL-17RB recruits the ubiquitin ligase tripartite motif containing 56 to add lysine-63-linked ubiquitin chains to lysine-470 of IL-17RB, which further assembles NF-κB activator 1 (ACT1) and other factors to propagate downstream oncogenic signaling. Consequentially, IL-17RB mutants with substitution at either tyrosine-447 or lysine-470 lose their oncogenic activity. Treatment with a peptide consisting of amino acids 403 to 416 of IL-17RB blocks MLK4 binding, tyrosine-477 phosphorylation, and lysine-470 ubiquitination in vivo, thereby inhibiting tumorigenesis and metastasis and prolonging the life span of mice bearing pancreatic tumors. These results establish a clear pathway of how proximal signaling of IL-17RB occurs and provides insight into how this pathway provides a therapeutic target for pancreatic cancer.

Targeting interleukin-17 receptor B enhances gemcitabine sensitivity through downregulation of mucins in pancreatic cancer.

Pancreatic cancer is the fourth leading cause of death worldwide due to its poorest prognoses with a 7% 5-year survival rate. Eighty percent of pancreatic cancer patients relapse after chemotherapy and develop early metastasis and drug resistance. Resistance to nucleoside analog gemcitabine frequently used in first-line therapy is an urgent issue in pancreatic cancer treatment. Expression of mucin (MUC) glycoproteins has been shown to enhance chemoresistance via increased cell stemness. Here we show interlukine-17 receptor B (IL-17RB) expression is positively correlated with MUC1 and MUC4 expression in pancreatic cancer cells and tumor tissue. Moreover, IL-17RB transcriptionally up-regulates expression of MUC1 and MUC4 to enhance cancer stem-like properties and resistance to gemcitabine. These results suggest IL-17RB can be a potential target for pancreatic cancer therapy. Indeed, treatment with IL-17RB-neutralizing antibody has a synergistic effect in combination with gemcitabine for killing pancreatic cancer cells. Altogether, these findings provide feasible applications for IL-17RB-targeting therapy in pancreatic cancer treatment.

DNA damages induced by trans, trans-2,4-decadienal (tt-DDE), a component of cooking oil fume, in human bronchial epithelial cells.

Epidemiological studies have demonstrated that cooking oil fumes (COF) are an environmental risk factor for the development of lung adenocarcinoma among nonsmoking females in Taiwan. Aside from polycyclic aromatic hydrocarbons, aldehydes, especially trans, trans-2,4-decadienal (tt-DDE) are found to be abundant in COF. Although there is indication that tt-DDE induces DNA damage, the precise role of tt-DDE in the induction of DNA damage in lung cells is still not clear. When we assessed DNA breaks with the Comet assay, we found that the DNA breaks induced by 1 muM tt-DDE in human bronchial epithelial cells (BEAS-2B) could be significantly reduced by antioxidants, suggesting that oxidative stress was involved. Indeed, when tt-DDE-treated cells were coincubated with endonuclease III/formamidopyrimidine-DNA glycosylase or with nuclear extract (NE), an enhancement of DNA breaks was observed at 1 hr after tt-DDE exposure. Furthermore, when NE was incubated with an antibody against 8-oxoguanine DNA glycosylase (anti-OGG1), a reduction in tt-DDE/NE-induced DNA breaks could be demonstrated. Since OGG1 is a specific repair enzyme for 8-oxo-deoxyguanosine (8-oxo-dG), these findings indicated that 8-oxo-dG was involved. On the other hand, when NE was incubated with antibodies against nucleotide excision repair enzymes, there was a significant reduction in tt-DDE/NE-induced DNA breaks at 4 hr after tt-DDE treatment. These observations indicate that, in addition to early oxidative DNA damage, nonoxidative DNA damage such as bulky adduct formation, was also induced by tt-DDE. Our study further affirms that tt-DDE is genotoxic to human lung cells and can increase carcinogenic risk.