Squamous Cell Carcinoma Tissue Specimens Research Articles

Survival-associated N6-adenosine methyltransferase signatures in lung squamous cell carcinoma and clinical verification

BackgroundN6-methyladenine (m6A) is the most common modification of mRNA and IncRNA in higher organisms. m6A has been confirmed to be related to the formation and progression of tumors and m6A-related genes can be used as prognostic biomarkers in a variety of tumors. However, there have been no similar studies on lung squamous cell carcinoma. The main purpose of this study was aimed to explore the differential expression of m6A-related genes in lung squamous cell carcinoma tissues and its relationship with patient clinical prognosis.MethodsWe integrated three m6A writers that catalyze the methylation of adenine on mRNA molecules. The training set including 501 patients with LUSC was collected from The Cancer Genome Atlas (TCGA) database and the test set including 181 patients with LUSC was collected from the Gene Expression Omnibus (GEO) database. Based on the expression level of the m6A methylase gene, we established a tumor subgroup and risk-prognosis model to quantify the risk index and long-term patient prognosis, which were confirmed by principal component analysis (PCA) and receiver operating characteristic (ROC) curve analysis. After lung squamous cell carcinoma tissue specimens were obtained during surgery, immunohistochemistry (IHC) was used to verify the results in vitro.ResultsThe results of the study showed that the expression of the three m6A methylases in tumor tissues and normal tissues was significantly different (P < 0.05). The survival-prognostic model based on METTL3 gene expression showed better predictive performance (AUC: 0.706). Patients in the high-risk and low-risk groups exhibited significant differences in terms of survival time and 5-year and 10-year survival rates. Immunohistochemistry revealed that patients with high METTL3 expression exhibited a longer survival time than those with low METTL3 expression.ConclusionsOur study showed that the molecular phenotype based on the expression of METTL3 may be an independent risk factor affecting the prognosis of lung squamous cell carcinoma. These findings not only prove the important role of m6A methylase in lung squamous cell carcinoma, but are also expected to provide more accurate prognostic assessment and individualized treatment for patients with lung squamous cell carcinoma.

N &lt;sup&gt;6&lt;/sup&gt;-Adenosine Methyltransferase METTL3 is a Positive Prognostic Biomarker in Lung Squamous Cell Carcinoma

Background: N6-methyladenine is the most common modification of mRNA and IncRNA in higher organisms. m6A has been confirmed to be related to the formation and progression of tumors and m6A-related genes can be used as prognostic biomarkers in a variety of tumors. However, there have been no similar studies on lung squamous cell carcinoma. The main purpose of this study was aimed to explore the differential expression of m6A-related genes in lung squamous cell carcinoma tissues and its relationship with patient clinical prognosis. Methods: We integrated three m6A writers that catalyze the methylation of adenine on mRNA molecules. The training set including 501 patients with LUSC was collected from The Cancer Genome Atlas database and the test set including 181 patients with LUSC was collected from the Gene Expression Omnibus database. Based on the expression level of the m6A methylase gene, we established a tumor subgroup and risk-prognosis model to quantify the risk index and long-term patient prognosis, which were confirmed by principal component analysis and receiver operating characteristic curve analysis. After lung squamous cell carcinoma tissue specimens were obtained during surgery, immunohistochemistry was used to verify the results in vitro. Results: The results of the study showed that the expression of the three m6A methylases in tumor tissues and normal tissues was significantly different (P<0.05). The survival-prognostic model based on METTL3 gene expression showed better predictive performance (AUC: 0.706). Patients in the high-risk and low-risk groups exhibited significant differences in terms of survival time and 5-year and 10-year survival rates. Immunohistochemistry revealed that patients with high METTL3 expression exhibited a longer survival time than those with low METTL3 expression. Conclusions: Our study showed that the expression level of m 6 A methylase in tumor tissues was much higher than that in normal tissues. At the same time, the molecular phenotype based on the expression of METTL3 may be an independent risk factor affecting the prognosis of lung squamous cell carcinoma. These findings not only prove the important role of m6A methylase in lung squamous cell carcinoma, but are also expected to provide more accurate prognostic assessment and individualized treatment for patients with lung squamous cell carcinoma. Funding: This study was supported by the Taishan Scholar foundation (No. tshw201502061). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: The Affiliated Hospital of Qingdao University Institutional Review Board approved this study, and all study subjects provided informed consent..

Inhibition of EGFR Abrogates Radioresistance in Human Oral Cancer by Inhibiting the Endoplasmic Reticulum Stress Chaperone GRP78

Combined radiotherapy and EGFR-targeted therapy regimen is preferred for patients with advanced oral cancer because it improves local cancer control and overall survival (OS) than radiotherapy alone. However, 50% of patients still develop local recurrence because of radioresistance. And some tumors showed radioresistance after inhibiting EGFR and the downstream signalling ERK, AKT,etc. Therefore, this study sought to identify new targets involved in resistance to EGFR-targeted therapy combined with radiotherapy.Endoplasmic reticulum stress (ERS) plays an important role in the regulation of radio- and chemoresistance in many malignancies. In this study, we investigated the role of EGFR and ERS signaling in the radioresistance of oral cancer. EGFR and ERS signalling pathway activation in KBoral carcinoma cells and the constructed corresponding radioresistant KBR cells after irradiation were detected by western blotting and real-time quantitative PCR. The radioresistance ofEGF-EGFR was detected using the colony formation assay. The cell apoptosis was detected by flow cytometry. And Cell proliferation was analysed using a Cell Counting Kit-8 (CCK-8) manufacturer’s protocol. DNA double-strand breaks and autophagy was detected by immunofluorescence. Immunohistochemistry was performed on 51 oral squamous cell carcinoma tissue specimens. The correlation between GRP78 and the prognosis of oral cancer patients was analysed using the Kaplan-Meier method. Herein, we constructed radioresistant oral cancer cells (KBR) and confirmed that EGF induced radioresistance, which could be reversed by silencing EGFR. Moreover, EGF activated radiation-induced ERS signalling PERK/ATF6/IRE1-GRP78 in oral cancer cells, whereas silencing EGFR inhibited the aforementioned functions. And EGF significantly increased the radiation-induced phosphorylation of ERK and AKT, while silencing PERK, IRE1α and ATF6 did not alter this effect. Consistent with others, ERS signalling in parallel with AKT/ERK in downstream of EGFR, and therefore may be targeted in amelioration of EGF-EGFR. In addition, silencing EGFR and the EGFR-targeted inhibitors cetuximab or nimotuzumab could inhibit radiation-induced DNA double-strand break (DSB) repair and autophagy, but increase radiation-induced cell apoptosis. The use of the ERS activators tunicamycin and thapsigargin could counteract this effect. Immunohistochemistry results showed that the ERS chaperone GRP78 was associated with poor prognosis of oral cancer (p < 0.05). These data indicate that inhibition of EGFR could reverse the radioresistance of oral cancer cells by inhibiting radiation-induced activation of ERSsignalling PERK/ATF6/IRE1-GRP78.

Upregulation of stem cell markers ALDH1A1 and OCT4 as potential biomarkers for the early detection of cervical carcinoma

Previous studies have reported the upregulation of stem cell biomarkers that are associated with tumorigenesis, in particular with cancer infiltration, recurrence and metastasis. Infection by human papilloma virus (HPV) is the main etiopathological factor of cervical carcinogenesis, but the expression of stem cell markers in cervical carcinoma and HPV infection have yet to be investigated so far. A total of 94 cases of fresh cervical tissues, 116 cases of paraffin-embedded cervical specimens and 72 cases of peripheral blood samples were collected from Uighur women who were either diagnosed with cervical squamous cell carcinoma (SCC) or cervical intraepithelial neoplasia (CIN) II–III, or from healthy subjects (negative controls, NC). HPV infection was detected in tissue DNA by polymerase chain reaction (PCR) with a HPV genotyping kit. The mRNA expression levels of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), nanog homeobox (NANOG), POU class 5 homeobox 1 (OCT4), SRY-box 2 (SOX2) and twist family BHLH transcription factor 1 (Twist1) were determined using reverse transcription-quantitative PCR (RT-qPCR). Histological analysis was performed in order to examine the protein expression of ALDH1A1 and OCT4 in paraffin-embedded tissue specimens by immunohistochemical staining and the plasma levels of those two proteins was measured by ELISA. RT-qPCR analysis indicated a significant increase in the mRNA expression of ALDH1A1 and OCT4 in CIN II–III and SCC tissue specimens compared with NC (P<0.05). Although the expression levels of NANOG, SOX2 and Twist1 were significantly higher in SCC compared with NC (P<0.05), no significant difference was revealed in CIN II–III tissues compared with SCC or NC (P>0.05). Subsequent analysis by immunohistochemistry staining confirmed that the upregulation of ALDH1A1 and OCT4 was also significantly increased in SCC and CIN II–III compared with controls at the protein level. Notably, ELISA analysis detected significantly higher levels of ALDH1A1 and OCT4 in the peripheral blood (plasma) of patients with SCC compared with healthy subjects. The upregulation of stem cell markers ALDH1A1 and OCT4 in cervical carcinoma and its precursor lesions, in particular in the peripheral blood, indicates that ALDH1A1 and OCT4 may serve as biomarkers for the early detection of cervical carcinoma or for the monitoring of treatment of patients.

The Role of GOLPH3L in the Prognosis and NACT response in Cervical Cancer.

Background: We previously reported GOLPH3L is a novel oncogene associated with ovarian cancer. The role of GOLPH3L in cervical cancer and its cellular functions has not been determined. This study investigated clinical significance of GOLPH3L and potential proteins and pathways associated with GOLPH3L in cervical squamous cell carcinoma.Methods: Immunohistochemistry and western blot were used to examine the expression of GOLPH3L in cervical squamous cell carcinoma tissue specimens and adjacent non-cancerous tissues. The clinical and prognostic significance of GOLPH3L expression was statistically analyzed. Cell proliferation rate, cell cycle progression, apoptosis and cisplatin response in GOLPH3L silenced SiHa and HeLa cells were also examined. Phospho-antibody array was used to identify changes in protein phosphorylation and the corresponding signaling pathways associated with these changes.Results: GOLPH3L overexpressed in cervical cancer tissue specimens compared with normal adjacent non-cancerous tissues. Increased GOLPH3L expression was associated with FIGO staging (P=0.033), cervical stromal invasion (P=0.037), cervical canal stromal invasion (P=0.027), lymph node metastasis (P=0.016) and positive surgical margins (P=0.015). Patients with lower expression of GOLPH3L demonstrated longer progression-free survival and overall survival compared with those with higher expression. The tissue samples from patients who poorly responded to neoadjuvant chemotherapy (NACT) exhibited increased GOLPH3L expression levels compared with tissue samples from patients who achieved a pathologic complete response (pCR). Patients with lower GOLPH3L expression level, poorer tumor differentiation, shorter NACT treatment intervals and smaller tumor sizes were more likely to achieve a pCR after NACT. Knockdown GOLPH3L in cells was associated with an induction of cell cycle arrest, increased apoptosis and cisplatin sensitivity, and a reduction in cellular viability. Phospho-antibody array suggested GOLPH3L plays a role in mediating cell cycle arrest.Conclusions: This study provides a potential biomarker for predicting prognosis and NACT response in patients with cervical squamous cell carcinoma. The functional role of GOLPH3L in cervical cancer merits further investigation.

CXCR7 expression correlates with tumor depth in cutaneous squamous cell carcinoma skin lesions and promotes tumor cell survival through ERK activation.

The chemokine receptor CXCR7 has been demonstrated to be involved in the development of certain cancers, but its role in cutaneous squamous cell carcinoma (SCC) has not been previously investigated. We seek to determine whether CXCR7 is expressed in human cutaneous SCC skin lesions and SCC cell lines. In addition, we evaluate whether CXCR7 plays a role in SCC cell proliferation, survival and migration and which signalling pathways are involved. Using quantitative RT-PCR to analyse the mRNA expression of 19 different chemokine receptors, we found that CXCR7 was much more highly expressed compared to other chemokine receptors in cutaneous SCC cell lines (HSC-1 and HSC-5). On immunohistochemical staining, CXCR7 was found to be expressed in 70% (28 of 40) of human cutaneous SCC tissue specimens, and its expression correlated with tumor depth >4mm and cancer stage ≥II. CXCR7 but not CXCR4 protein was expressed on the surface of HSC-1 and HSC-5 cells by flow cytometry. Activation of the CXCR7 receptor by CXCL12 promoted survival of HSC-1 and HSC-5 cells through the ERK pathway, but had no significant effect on cell proliferation or migration. In summary, our findings indicate that CXCR7 is frequently expressed in cutaneous SCC skin lesions and its expression correlates with tumor depth and cancer stage. CXCR7 is the predominant chemokine receptor expressed in SCC cell lines, and activation of CXCR7 by CXCL12 promotes survival of SCC cells through the ERK pathway. These findings provide new insights into the significance of CXCR7 in the pathophysiology of SCC.

Expression of ZNF396 in basal cell carcinoma

Zfp191 represses differentiation and keeps various cells in the stem/progenitor stage. Here, we report that a Zfp191 homolog protein, ZNF396, is expressed in basal cell carcinoma (BCC) and possibly represses the expression of a Notch system effector molecule, Hes1 (hairy and enhancer of split-1), and prevents BCC cells from undergoing Notch-mediated squamous cell differentiation. ZNF396 immunoreactivity was found in the nucleus of 35 of 38 cutaneous BCC and 4 of 74 squamous cell carcinoma tissue specimens. In non-tumorous epidermal tissues, ZNF396 immunoreactivity was restricted in basal cells. siRNA-mediated silencing of ZNF396 induced the expression of Notch2, Hes1, and involucrin in cultured BCC cells. Finally, we found that siRNA-mediated silencing of ZNF396 gene inhibited the proliferation of TE354.T basal cell carcinoma cells. ZNF396 might repress Notch-Hes1 signaling axis and prevent tumor cells from undergoing squamous differentiation in BCC.

Extracellular heat shock protein A9 is a novel interaction partner of podoplanin in oral squamous cell carcinoma cells

In previous studies, we have shown several lines of evidence that podoplanin (PDPN) plays an important role in cell adhesion via its association with extracellular components in neoplastic conditions, though there has been no trial to search for PDPN-interaction molecules in the extracellular milieu. To screen for those molecules, we performed proteomics-based analysis using liquid chromatography-tandem mass spectrometry followed by co-immunoprecipitation for PDPN in ZK-1, an oral squamous cell carcinoma (SCC) cell system whose cell membrane molecules were cross-linked with each other in their extracellular compartments, and we identified heat shock protein (HSP) A9 as one of the extracellular PDPN bound molecules. Effects of transient PDPN knockdown by siRNA in ZK-1 were also comparatively examined for cellular behaviors in terms of HSPA9 expression and secretion. Finally, HSPA9 expression modes were immunohistochemically visualized in oral SCC tissue specimens. HSPA9 was secreted from ZK-1 cells, and the expression and secretion levels of HSPA9 gene and protein were well coordinated with those of PDPN. Immunohistochemically, HSPA9 and PDPN were co-localized in ZK-1 cells and oral SCC foci, especially in the peripheral zone. In conclusion, the results indicate that HSPA9 secreted by oral SCC cells interacts with PDPN on their cell surface in an autocrine manner and regulates their growth and invasiveness.

Overexpression of RNA helicase p68 protein in cutaneous squamous cell carcinoma

RNA helicase p68 is a prototypic DEAD-box RNA helicase. Recent studies indicate that p68 plays important role in cancer development and progression. However, the role of p68 protein expression in cutaneous squamous cell carcinoma (SCC) remains unknown. To elucidate the expression of p68 protein in cutaneous SCC. The level of p68 protein was examined by double immunofluorescent staining in 24 samples of human cutaneous SCC tissue specimens and their adjacent tissues and in 6 normal foreskin samples to compare the expression of p68 with that of Ki-67. Overexpression of p68 protein was seen in all 24 SCC cases (100%), whereas very low expression of p68 was detected in normal foreskin. Moreover, p68 protein expression was higher in cases of cutaneous SCC with metastasis than in cases without metastasis. Additionally, p68 had a similar expression pattern to that of Ki-67. The high frequency of p68 expression in cutaneous SCC indicates that p68 might be involved in the development and progression of cutaneous SCC.

Expressions of phosphatidylinositol 3 kinase and phosphorylated Akt in condyloma acuminatum and cervical squamous cell carcinoma

Objective To investigate the roles of phosphatidylinositol 3 kinase (PI3K) and phosphorylated Akt (P-Akt) in the pathogenesis of cervical squamous cell carcinoma and condyloma acuminatum.Methods Immunohistochemistry and Western blot were used to detect the expressions of PI3K and P-Akt in tissue specimens from the lesions of 30 cases of cervical squamous cell carcinoma,30 cases of condyloma acuminatum and the prepuce of 15 normal human controls.The average optical density and gray scale values were calculated and analyzed by t test and F test respectively.Results The expressions of PI3K and P-Akt were observed in only the basal layer of the epidermis of control specimens,but in the whole epidermis of condyloma acuminatum tissue specimens.Cervical squamous cell carcinoma tissue specimens displayed a stronger expression of PI3K and P-Akt compared with the control and condyloma acuminatum tissue specimens.As immunohistochemistry revealed,the average absorbance value for PI3K and P-Akt was 0.28 ±0.05 and 0.20 ± 0.07 respectively in cervical squamous cell carcinoma tissue specimens,0.22 ± 0.04 and 0.17 ± 0.03 respectively in condyloma acuminatum tissue specimens,and 0.16 ± 0.04 and 0.10 ± 0.02 respectively in the control tissue specimens; significant differences were observed in the expressions of PI3K and P-Akt among the three groups of tissue specimens (F =44.87,20.64,respectively,both P ＜ 0.01 ).The results of Western blot were consistent with those of immunohistochemistry,and there was a significant difference in the gray scale value for PI3K and P-Akt between cervical squamous cell carcinoma,condyloma acuminatum and control tissue specimens (3.48 ± 0.48 vs.1.99 ± 0.11 vs.1.00 ± 0.03,F=354.83,P＜ 0.01; 3.33 ± 0.26 vs.1.96 ± 0.11 vs.1.00 ± 0.03,F=302.33,P＜ 0.01 ).Conclusions The PI3K/Akt signaling pathway is abnormally activated in condyloma acuminatum and cervical squamous cell carcinoma,and human papilloma virus may cause the abnormal proliferation of infected epithelium likely by affecting the upregnlated expression of PI3K/P-Akt. Key words: Carcinoma, squamous cell; Condylomata acuminata; l-Phosphatidylinositol 3-kinase; Proto-oncogene proteins c-akt

Association between human papillomavirus infection and laryngeal squamous cell carcinoma

The aim of this study was to compare the prevalence of human papillomavirus (HPV) infection in laryngeal squamous cell carcinoma using two methods: PCR-DNA enzyme immunoassay (PCR/DEIA) and immunohistochemistry (IHC) for detection of HPV in specimens of laryngeal squamous cell carcinoma and to correlate the presence of HPV with the epidemiological and clinicopathological features of recurrence and survival. HPV DNA was amplified from 93 paraffin-embedded laryngeal squamous cell carcinoma tissue specimens by the short PCR fragment (SPF 10) primer set using PCR/DNA method. HPV detection using monoclonal anti-human papilloma virus antibodies Clone K1H8 for IHC reaction was performed on 130 specimens. HPV was identified in 35.5% of patients with laryngeal squamous cell carcinoma using PCR/DEIA and 27.7% using IHC. There was no statistically significant association between the presence of HPV and the epidemiological and clinicopathological features and recurrence. There was no statistically significant association between the presence of HPV and overall survival nor disease specific survival. Statistically significant correlation between HPV detection using PCR/DEIA technique and IHC technique was found. The presence of HPV infection in 27.7% and 38.9% of the patients suggests a possible role in the etiology of laryngeal squamous cell carcinoma. The SPF(10) PCR/DEIA technique is the most accurate method for detection of HPV in laryngeal squamous cell carcinoma.

Elevated salivary endothelin levels in oral cancer patients—A pilot study

The analysis of saliva has been proposed as a potentially rapid, non-invasive method to monitor and diagnose patients with oral disease. In this study we measured salivary endothelin-1 (ET-1) levels in patients diagnosed with oral squamous cell carcinoma (SCC) prior to treatment. We demonstrate significantly elevated salivary ET-1 levels in the oral SCC group (4.37+/-1.35pg/ml), relative to the control group (1.16+/-0.29pg/ml). ET-1 and ET-1 mRNA were also measured in oral SCC tissue specimens and compared to normal oral epithelial controls. The concentration of ET-1 in the oral SCC specimens was 17.87+/-4.0pg/ml and in the normal epithelial controls the concentration of ET-1 was 5.43+/-2.5pg/ml. ET-1 mRNA was significantly overexpressed in 80% (8/10) of the oral SCC specimens. Our results demonstrate the potential utility of salivary analysis for ET-1 levels to monitor patients at risk for oral SCC.