Prognostic implications of the nuclear localization of Y-box-binding protein-1 and CXCR4 expression in ovarian cancer: their correlation with activated Akt, LRP/MVP and P-glycoprotein expression

The nuclear localization of Y-box-binding protein-1 (YB-1) is known to be a poor prognostic factor in several human malignancies, including ovarian carcinoma. Following on from our basic study dealing with microarray analyses of YB-1-associated gene expression in ovarian cancer cells, we examined whether nuclear localization of YB-1 is associated with the expression of CXCR4, a vault protein named lung resistance-related vault protein (LRP/MVP), phosphorylated Akt (p-Akt) or P-glycoprotein (P-gp) in human ovarian carcinoma. Fifty-three surgically resected ovarian carcinomas treated with paclitaxel and carboplatin were examined immunohistochemically for nuclear YB-1 expression and intrinsic expression of p-Akt, P-gp, LRP/MVP and CXCR4. Nuclear expression of YB-1 demonstrated significant correlation with p-Akt, P-gp and LRP expression, but no relationship with CXCR4 expression. By multivariate analysis, only YB-1 nuclear expression and CXCR4 expression were independent prognostic factors with regard to overall survival. These results indicate that YB-1 nuclear expression and CXCR4 expression are important prognostic factors in ovarian carcinoma.     

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer

BackgroundRecent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death–ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC.Patients and methodsThe expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry.ResultsExpression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients.ConclusionsHigh expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.     

The transcription factor DEC1 (BHLHE40/STRA13/SHARP-2) is negatively associated with TNM stage in non-small-cell lung cancer and inhibits the proliferation through cyclin D1 in A549 and BE1 cells

The objective of the current study was to investigate the expression pattern and clinicopathological significance of differentiated embryo–chondrocyte-expressed gene 1 (DEC1) in patients with non-small-cell lung cancer (NSCLC). In 118 archived NSCLC tissues, the positive rate of DEC1 was reduced in human lung cancer samples (36/118, 30.5 %) compared with adjacent normal lung tissues (106/118, 89.8 %), as measured by immunohistochemical staining. Loss of DEC1 was correlated with poor differentiation (p?=?0.005) and high p-TNM stage (p?=?0.002). Consistently, downregulation of DEC1 by siRNA knockdown promoted the growth and colony formation in the A549 lung cancer cell line, and overexpression of DEC1 inhibited the growth and colony formation in the BE1 lung cancer cell line. In addition, a significant negative correlation was found between DEC1 and cyclin D1 (p?=?0.014) in 118 cases of NSCLC. Knockdown of DEC1 resulted in the upregulation of cyclin D1, and overexpression of DEC1 led to the downregulation of cyclin D1. Together with the observation that DEC1 bound directly to the promoter region of cyclin D1 in A549 cells, these results indicate that loss of DEC1 may promote tumor progression in NSCLC through upregulation of cyclin D1, and DEC1 might serve as a novel therapeutic target of NSCLC.     

Expression of HER2 and estrogen receptor alpha depends upon nuclear localization of Y-box binding protein-1 in human breast cancers

In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER)alpha, ER beta, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ER alpha expression in ER alpha-positive, but not ER alpha-negative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ER alpha (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt-dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ER alpha.     

The functional status of DNA repair pathways determines the sensitization effect to cisplatin in non-small cell lung cancer cells

Purpose

Cisplatin can cause a variety of DNA crosslink lesions including intra-strand and inter-strand crosslinks (ICLs), which are associated with the sensitivity of cancer cells to cisplatin. Here, we aimed to assess the contribution of the Fanconi anemia (FA), homologous recombination (HR) and nucleotide excision repair (NER) pathways to cisplatin resistance in non-small cell lung cancer (NSCLC)-derived cells.

Methods

The expression of FA, HR and NER pathway-associated genes was assessed by RT-qPCR and Western blotting. siRNAs were used to knock down the expression of these genes. CCK-8 and flow cytometry assays were used to assess the viability and apoptotic rate of NSCLC-derived cells, respectively. Immunofluorescence and alkaline comet assays were used to assess the repair of ICLs.

Results

We found that acquired cisplatin-resistant NSCLC-derived A549/DR cells exhibited markedly enhanced FA and HR repair pathway capacities compared to its parental A549 cells and another independent NSCLC-derived cell line, Calu-1, which possesses a moderate innate resistance to cisplatin. siRNA-mediated silencing of the FA-associated genes FANCL and RAD18 and the HR-associated genes BRCA1 and BRCA2 significantly potentiated the sensitivity of A549/DR cells to cisplatin compared to A549 and Calu-1 cells, suggesting that the acquired cisplatin resistance in A549/DR cells may be attributed to enhanced FA and HR pathway capacities responsible for ICL repair. Although we found that expression knockdown of the NER-associated genes XPA and ERCC1 sensitized the three NSCLC-derived cell lines to cisplatin, the sensitization effect was more significant in Calu-1 cells than in A549 and A549/DR cells, implying that the innate cisplatin resistance in Calu-1 cells may result from an increased NER activity.

Conclusions

Our results indicate that the functional status of DNA repair pathways determine the sensitivity of NSCLC cells to cisplatin. Direct targeting of the pathway that is involved in cisplatin resistance may be an effective strategy to surmount cisplatin resistance in NSCLC.

     

Clinical significance of IGF1R expression in non-small-cell lung cancer

BackgroundThe purpose of the current study was to clarify the clinical role of insulin-like growth factor receptor-1 (IGF1R) in NSCLC.Patients and MethodsTumor specimens were collected from 285 patients who underwent complete resection for adenocarcinoma (AD, n = 182), squamous cell carcinoma (SCC, n = 77), and other histologic types of cancer (n = 26) of the lung. The expression of IGF1R and Ki-67 was evaluated by immunohistochemical (IHC) analysis.ResultsPositive expression of IGF1R was detected in 87 (30.5%) of 285 cases, of which 43 (23.6%) of 182 cases were AD, 36 (46.8%) of 77 cases were SCC, and 8 (30.8%) of 26 cases were other histologic types (SCC vs. AD, p < .001; SCC vs. non-SCC, p < .001). Positive IGF1R expression was also identified in 20 (44.4%) and 67 (27.9%) of the patients with and without recurrence, respectively (p = .027). Multivariate logistic regression models indicated that positive staining for IGF1R expression was an independent factor in AD associated with tumor recurrence (p = .040) but not in NSCLC, SCC, and other types of cancer. A positive IGF1R expression tended to demonstrate a poor disease-free survival (DFS) in NSCLC according to the Kaplan-Meier DFS curves (p = .053). The tumors showing a positive expression of IGF1R were observed more frequently in tumors with a positive expression of Ki-67 than in the tumors with a negative expression of Ki-67 (p = .010).ConclusionIGF1R expression was associated with reduced DFS correlating with postoperative recurrence. In addition, a significant relationship was also observed between IGF1R and Ki-67 expression in NSCLC. However, in subgroup analysis, a significant correlation was not observed. IGF1R expression predicts postoperative recurrence in patients with AD, but not in those with non-AD of NSCLC.     

miR-1238 inhibits cell proliferation by targeting LHX2 in non-small cell lung cancer

In human cancers, dysregulated expression of LIM-homeobox gene 2 (LHX2) and downregulation of miR-1238 has been reported separately. However, the relationship between them remains unclear. We investigated the functional contribution of miR-1238 to the regulation of LHX2 in non-small cell lung cancer (NSCLC). Here, computational algorithms predicted that the 3′-untranslated region (3′-UTR) of LHX2 is a target of miR-1238. Luciferase assays validated that miR-1238 directly bound to 3′-UTR of LHX2. qRT-PCR and western blot analyses further confirmed that overexpression of miR-1238 mimic in NSCLC A549 and LTEP-α-2 cells inhibited endogenous expression of LHX2 mRNA and protein. Moreover, ectopic expression of miR-1238 in NSCLC A549 and LTEP-α-2 cells suppressed cellular viability and proliferation. siRNA-induced knockdown of LHX2 copied the phenotype of miR-1238 overexpression in NSCLC A549 and LTEP-α-2 cells and LHX2 knockdown inhibited cell cycle. In addition, miR-1238 expression was frequently decreased in human NSCLC tissues and reversely correlated with LHX2 expression, which was increased in NSCLC tissues. Collectively, our findings demonstrate that miR-1238 inhibit the proliferation of NSCLC cells at least partly via repression of LHX2, shedding light on the mechanistic interaction of miR-1238 and LHX2 in NSCLC carcinogenesis. Furthermore, our data suggest that expression of miR-1238 could be a promising therapeutic strategy for NSCLC treatment.     

Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer

BackgroundUpregulated expression and aberrant activation of the epidermal growth-factor receptor (EGFR) are found in lung cancer, making EGFR a relevant target for non-small-cell lung cancer (NSCLC). Treatment with anti-EGFR monoclonal antibodies (mAbs) is associated with modest improvement in overall survival in patients with squamous cell lung cancer (SqCLC) who have a significant unmet need for effective treatment options. While there is evidence that using EGFR gene copy number, EGFR mutation, and EGFR protein expression as biomarkers can help select patients who respond to treatment, it is important to consider biomarkers for response in patients treated with combination therapies that include EGFR mAbs.DesignRandomized trials of EGFR-directed mAbs cetuximab and necitumumab in combination with chemotherapy, immunotherapy, or antiangiogenic therapy in patients with advanced NSCLC, including SqCLC, were searched in the literature. Results of associations of potential biomarkers and outcomes were summarized.ResultsData from phase III clinical trials indicate that patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein (H-score of ≥200) and/or gene copy numbers of EGFR (e.g. ≥40% cells with ≥4 EGFR copies as detected by fluorescence in situ hybridization; gene amplification in ≥10% of analyzed cells) derive greater therapeutic benefits from EGFR-directed mAbs. Biomarker data are limited for EGFR mAbs used in combination with immunotherapy and are absent when used in combination with antiangiogenic agents.ConclusionsTherapy with EGFR-directed mAbs in combination with chemotherapy is associated with greater clinical benefits in patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein and/or have increased EGFR gene copy number. These data support validating the role of these as biomarkers to identify those patients who derive the greatest clinical benefit from EGFR mAb therapy. However, data on biomarkers for EGFR-directed mAbs combined with immunotherapy or antiangiogenic agents remain limited.     

High coexpression of both insulin-like growth factor receptor-1 (IGFR-1) and epidermal growth factor receptor (EGFR) is associated with shorter disease-free survival in resected non-small-cell lung cancer patients

BackgroundInsulin-like growth factor receptor-1 (IGFR-1) represents a novel molecular target in non-small-cell lung cancer (NSCLC). IGFR-1 and epidermal growth factor receptor (EGFR) activation is essential to mediate tumor cell survival, proliferation and invasion. We explored the correlation between IGFR-1 and EGFR, their relationship with clinicopathological parameters and their impact on outcome in resected stage I–III NSCLC patients.Patients and methodsTumors from 125 surgical NSCLC patients were evaluated for IGFR-1 and EGFR expression by immunohistochemistry. Kaplan–Meier estimates of survival and time to recurrence were calculated for clinical variables and biologic markers using the Cox model for multivariate analysis.ResultsIGFR-1 protein overexpression was detected in 36.0% of NSCLC patients and was associated with larger tumor size (P = 0.04) but not with other clinical or biological characteristics. EGFR protein overexpression was observed in 55.2% of NSCLC, more frequently in squamous cell carcinoma (SCC) than non-SCC (63.7% versus 36.3%, χ² = 9.8, P = 0.001). IGFR-1 protein expression was associated with EGFR protein expression (P = 0.03). At the multivariate analysis, high coexpression of both IGFR-1 and EGFR was a significant prognostic factor of worse disease-free survival (DFS) (hazard ratio 2.51, P = 0.01).ConclusionA statistically significant association was observed between high coexpression of both IGFR-1 and EGFR and worse DFS in early NSCLC patients.     

DLC-1过表达对非小细胞肺癌细胞增殖和侵袭的影响

目的 探讨肝癌缺失基因1(deleted in liver cancer-1,DLC-1)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖和侵袭的影响及其作用机制.方法 构建pcDNA3.1-DLC-1重组质粒并转染至人非小细胞肺癌A549和Calu-6细胞,采用Western...     

Expression of insulin-like growth factor 1 receptor (IGF-1R) predicts poor responses to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer patients harboring activating EGFR mutations

ObjectivesExpression of insulin-like growth factor 1 receptor (IGF-1R) in non-small cell lung cancer (NSCLC) is associated with poor prognosis. The IGF-1R pathway activates downstream targets that bypass dependency in signals from the epidermal growth factor receptor (EGFR), which mediates resistance to EGFR tyrosine kinase inhibitors (TKIs). The aim of the present study was to determine the predictive role of IGF-1R expression in the response to EGFR-TKIs of NSCLC patients harboring activating EGFR mutations.Materials and methodsWe retrospectively studied 62 NSCLC patients who had activating EGFR mutations and received TKIs. Protein expression of IGF-1R, vascular endothelial growth factor (VEGF), and human epidermal growth factor receptor 2 (HER2) were measured by immunohistochemical staining. Univariate and multivariate analyses were performed to identify predictive factors associated with the responses to EGFR-TKIs. The relationship of progression-free survival (PFS) with IGF-1R expression and the presence of diabetes mellitus (DM) were examined.ResultsOf 62 EGFR mutation positive patients, 26 expressed IGF-1R, and 13 had DM. In the multivariate analysis, young age, squamous cell carcinoma, and IGF-1R expression were independently associated with a shorter PFS after treatment with EGFR-TKIs. Patients expressing IGF-1R showed a significantly shorter PFS in response to EGFR-TKIs compared with those lacking IGF-1R expression (9.1 vs. 20.1 months, p = 0.005). The 13 patients with DM were more likely to express IGF-1R (p = 0.001) and had shorter PFS times when treated with first-line EGFR-TKIs (7.6 vs. 18.6 months, p = 0.005), compared with those without DM.ConclusionIGF-1R expression was a negative predictive factor for a response to EGFR-TKIs in NSCLC patients harboring activating EGFR mutations. Moreover, patients with DM highly expressed IGF-1R in tumor tissues, which was associated with a poor response to first-line TKI therapy. Further studies aimed at overcoming EGFR-TKI resistance will need to also address IGF-1R pathways.     

Prognostic role of Twist, Slug, and Foxc2 expression in stage I non-small-cell lung cancer after curative resection

BackgroundLung cancer is the leading cause of cancer-related death in the world. Only about 60% of patients with stage I non–small-cell lung cancer (NSCLC) can be cured by surgery alone. Current clinical and molecular markers are inadequate prognosticators. We developed a 3-marker model that closely approximates survival probability of patients with stage I NSCLC.MethodsExpression of Twist, Slug, and Foxc2 was assessed by immunohistochemistry in tissue microarrays that contained paired tumor and peritumoral lung tissue from 137 patients who underwent surgical resection for stage I NSCLC. The prognostic value of Twist, Slug, and Foxc2, and the cumulative effects of the 3 markers on survival were evaluated.ResultsIncreased expression of Twist, Slug, and Foxc2 was observed in 38.0%, 18.2%, and 27.7% of primary tumors, respectively. Overexpression of Twist, Slug, and Foxc2 in stage I NSCLC was associated with a worse overall survival (P = .001, P = .002, P < .001, respectively) and correlated with a shorter recurrence-free survival (P < .001, P = .001, P < .001 respectively). The cumulative influence of these markers on outcome was analyzed; a combination of more than 2 positive markers was an independent predictor of recurrence-free and overall survival (P = .002 and P = .009, respectively).ConclusionsThe Twist/Slug/Foxc2 model is useful in predicting survival of stage I NSCLC and may be helpful in refining current treatment strategy.     

Insulin-like growth factor receptor 1 (IGF1R) expression and survival in surgically resected non-small-cell lung cancer (NSCLC) patients

BackgroundThe purpose of this study is to investigate the prognostic role of insulin-like growth factor receptor 1 (IGF1R) expression in surgically resected non-small-cell lung cancer (NSCLC).Patient characteristics and methodsThis retrospective study was conducted in 369 stage I–II–IIIA, surgically resected, NSCLC patients. Patients exposed to anti-epidermal growth factor receptor (EGFR) agents were excluded. IGF1R expression was evaluated by immunohistochemistry in tissue microarray sections.ResultsA positive IGF1R expression (score ≥ 100) was observed in 282 cases (76.4%) and was significantly associated with squamous cell histology (P = 0.04) and with grade III differentiation (P = 0.02). No difference in survival was observed between the positive and negative group when score 100 was used as cut-off for discriminating a positive versus a negative IGF1R result (52 versus 48 months, P = 0.99) or when median value of IGF1R expression was used (45 versus 55 months, P = 0.36). No difference in survival was observed between IGF1R-positive and -negative patients in a subgroup of stage I–II adenocarcinoma (n = 137) with known EGFR mutation and copy number status.ConclusionsIGF1R expression does not represent a prognostic factor in resected NSCLC patients. Patients with squamous cell carcinoma overexpress IGF1R more frequently than patients with nonsquamous histology, justifying the different sensitivity to anti-IGF1R agents observed in clinical trials.     

RNA干扰技术抑制A549细胞表皮生长因子受体表达的研究

目的探讨RNA干扰(RNAi)技术抑制非小细胞肺癌(NSCLC)细胞株A549细胞表皮生长因子受体(EGFR)表达后,对A549细胞生物学特性的影响。方法体外化学合成EGFR序列特异性双链RNA(dsRNA),用Lipofectamine 2000转染A549细胞,采用Western blot技术和流式细胞仪检测EGFR的表达,并观察A549细胞周期、集落形成、化疗敏感性等生物学特性的改变。结果序列特异性dsRNA-EGFR可显著抑制A549细胞EGFR表达;dsRNA-EGFR组细胞生长抑制率为85．0％,集落形成抑制率63．3％;细胞周期分析结果表明,dsRNA-EGFR组G0～G1期细胞数较对照组增加了12．7％,进入S期的细胞数较对照组减少了6．6％。转染dsRNA-EGFR后,可将A549细胞对顺铂的敏感性提高约4倍。结论dsRNA-EGFR可有效抑制A549细胞EGFR的表达,并将更多的细胞阻滞在G0～G1期,抑制细胞增生,显著增加细胞对顺铂的敏感性。RNAi可能为NSCLC的基因治疗提供新策略。