Radiotherapy diagnostic biomarkers in radioresistant human H460 lung cancer stem-like cells

Tumor cell radioresistance is a major contributor to radiotherapy failure, highlighting the importance of identifying predictive biomarkers for radioresistance. In this work, we established a radioresistant H460 (RR-H460) cell line from parental radiosensitive H460 lung cancer cells by exposure to fractionated radiation. The radiation-resistant, anti-apoptotic phenotype of RR-H460 cell lines was confirmed by their enhanced clonogenic survival and increased expression of the radioresistance genes Hsp90 and Her-3. RR-H460 cells displayed characteristics of cancer stem-like cells (CSCs), including induction of the surface marker CD44 and stem cell markers Nanog, Oct4, and Sox2. RR-H460 cells also exhibited sphere formation and malignant behavior, further supporting a CSC phenotype. Using proteomic analyses, we identified 8 proteins that were up-regulated in RR-H460 CSC lines and therefore potentially involved in radioresistance and CSC-related biological processes. Notably, 4 of these—PAI-2, NOMO2, KLC4, and PLOD3—have not been previously linked to radioresistance. Depletion of these individual genes sensitized RR-H460 cells to radiotoxicity and additively enhancing radiation-induced apoptosis. Our findings suggest the possibility of integrating molecular targeted therapy with radiotherapy as a strategy for resolving the radioresistance of lung tumors.     

Comparative Analysis of Oct4 in Different Histological Subtypes of Esophageal Squamous Cell Carcinomas in Different Clinical Conditions

Background: Esophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. It hasbeen hypothesized that Oct4 positive radioresistant stem cells may be responsible for tumor recurrence. Hence,we evaluated Oct4 expression in ESCC in pre-treatment, post neo-adjuvant residual and post-surgical recurrenttumours. Materials and Methods: Endoscopic mucosal biopsies were used to study Oct4 expression and theobservations were correlated with histological tumor grades, patient data and clinical background. Results: Allpatients presented with dysphagia with male predominance and a wide age range. Majority of the patients hadintake of mixed diet, history of alcohol and tobacco intake was documented in less than half of the patients. Oct4 expression was significantly higher in poorly differentiated (PDSCC) and basaloid (BSCC) subtypes than theother better differentiated tumor morphology. Oct4 was also expressed by adjoining esophageal mucosa showinglow grade dysplasia and basal cell hyperplasia (BCH). Biopsies in PDSCC and BSCC groups were more likelyto show a positive band for Oct4 by polymerase chain reaction (PCR). Dysplasia and BCH mucosa also showedOct4 positivity by PCR. All mucosal biopsies with normal morphology were negative for Oct4. Number of tissuesamples showing Oct4 positivity by PCR was higher than that by the conventional immunohistochemistry (p>0.05).Oct4 expression pattern correlated only with tumor grading, not with other parameters including the clinicalbackground or patient data. Conclusions: Our observations highlighted a possible role of Oct4 in identifyingputative cancer stem cells in ESCC pathobiology and response to treatment. The implications are either in vivoexistence of Oct4 positive putative cancer stem cells in ESCC or acquisition of cancer stem cell properties bytumor cells as a response to treatment given, resulting ultimately an uncontrolled cell proliferation and treatmentfailure.     

Selection of radioresistant tumor cells and presence of ALDH1 activity in vitro

Background

Tumor resistance to radiotherapy has been hypothesized to be mediated by a tumor subpopulation, called cancer stem cells (CSCs). Based on the proposed function of CSCs in radioresistance, we explored the cancer stem cell properties of cells selected for radioresistance phenotype.

Materials and methods

A549 and SK-BR-3 cells were radioselected with four single doses of 4 or 3 Gy in intervals of 10-12 days and used for colony formation assay and γ-H2AX foci formation assay. Expression of putative stem cell markers, i.e. Sox2, Oct4, ALDH1, and CD133 were analyzed using Western blotting. A549 and SK-BR-3 cells sorted based on their ALDH1 activity were analyzed in clonogenic survival assays.

Results

Radioselected A549 and SK-BR-3 cells (A549-R, SK-BR-3-R) showed increased radioresistance and A549-R cells presented enhanced repair of DNA-double strand breaks. PI3K inhibition significantly reduced radioresistance of A549-R cells. Cell line specific differences in the expression of the putative CSC markers Sox2 and Oct4 were observed when parental and radioselected cells were compared but could not be directly correlated to the radioresistant phenotype. However, enzyme activity of the putative stem cell marker ALDH1 showed a correlation to radioresistance.

Conclusions

Subpopulations of pooled radioresistant colonies, selected by various radiation exposures were analyzed for the presence of putative stem cell markers. Although the pattern of Sox2, Oct4, and CD133 expression was not generally associated with radioresistance, presence of ALDH1 seems to be indicative for subpopulations with increased radioresistance.     

Characterization of CD44 variant expression in head and neck squamous cell carcinomas

CD44 is a complex family of molecules, associated with aggressive malignancies and cancer stem cells. However, the role of CD44 variants in tumor progression and treatment resistance is not clear. In this study, the expression of CD44 and its variants was assessed in head and neck squamous cell carcinomas (HNSCC). Furthermore, subpopulations of cells expressing high amounts of CD44 variants were identified and characterized, for e.g., cell cycle phase and radioresistance. Results revealed high and homogenous CD44 and CD44v7 expression in four cell lines and CD44v4 and CD44v6 in three cell lines. CD44v3 was highly expressed in two cell lines, whereas CD44v5, CD44v7/8, CD44v10, CD133, and CD24 demonstrated no or moderate expression. Moreover, a subpopulation of very high CD44v4 expression was identified, which is independent of cell phase, demonstrating increased proliferation and radioresistance. In cell starvation experiments designed to enrich for cancer stem cells, a large population with dramatically increased expression of CD44, CD44v3, CD44v6, and CD44v7 was formed. Expression was independent of cell phase, and cells demonstrated increased radioresistance and migration rate. Our results demonstrate that the heterogeneity of tumor cells has important clinical implications for the treatment of HNSCC and that some of the CD44 variants may be associated with increased radioresistance. Highly expressed CD44 variants could make interesting candidates for selective cancer targeting.     

Cancer stem cell related markers of radioresistance in head and neck squamous cell carcinoma

Despite recent advances in understanding of the molecular pathogenesis and improvement of treatment techniques, locally advanced head and neck squamous cell carcinoma (HNSCC) remains associated with an unfavorable prognosis. Compelling evidence suggests that cancer stem cells (CSC) may cause tumor recurrence if they are not eradicated by current therapies as radiotherapy or radio-chemotherapy. Recent in vitro studies have demonstrated that CSCs may be protected from treatment-induced death by multiple intrinsic and extrinsic mechanisms. Therefore, early determination of CSC abundance in tumor biopsies prior-treatment and development of therapeutics, which specifically target CSCs, are promising strategies to optimize treatment. Here we provide evidence that aldehyde dehydrogenase (ALDH) activity is indicative for radioresistant HNSCC CSCs. Our study suggests that ALDH⁺ cells comprise a population that maintains its tumorigenic properties in vivo after irradiation and may provide tumor regrowth after therapy. We found that ALDH activity in HNSCC cells can be attributed, at least in part, to the ALDH1A3 isoform and inhibition of the ALDH1A3 expression by small interfering RNA (siRNA) decreases tumor cell radioresistance. The expression dynamic of ALDH1A3 upon irradiation by either induction or selection of the ALDH1A3 positive population correlates to in vivo curability, suggesting that changes in protein expression during radiotherapy are indicative for tumor radioresistance. Our data indicate that ALDH1A3⁺ HNSCC cells may contribute to tumor relapse after irradiation, and inhibition of this cell population might improve therapeutic response to radiotherapy.     

Cancer stem cell phenotype relates to radio-chemotherapy outcome in locally advanced squamous cell head-neck cancer

Background:

Cancer stem cells (CSCs) tend to repopulate malignant tumours during radiotherapy and, therefore, prolongation of the overall treatment time may result in radiotherapy failure. Thus, an estimate of the number of CSCs in tumour biopsies may prove most useful in predicting resistance to radiotherapy and a guide for development therapies aimed to eradicate a cancer cell population with effects on radiotherapy-related cancer regrowth.

Methods:

The CSC population was investigated semi-quantitatively in 74 locally advanced squamous cell head–neck cancers (HNSCC) from an equal number of patients, treated with accelerated platinum-based radiotherapy. A standard immunohistochemical technique and the CSC markers CD44, CD24, Oct4, integrin-β1 and aldehyde dehydrogenase isoform 1A1 (ALDHA1) was used, in parallel with the proliferation marker MIB-1. The results were correlated with the site of the tumour, the MIB-1 index, the tumour grade and stage, and prognosis.

Results:

The expression of CD44, CD24 and Oct4 were significantly associated with the MIB-1 proliferation index. In addition, the CD44 was linked with the better differentiated HNSCC. The CD44, Oct4 and integrin-β1 were all associated with poor prognosis but, in a multivariate analysis, the integrin-β1 had an independent statistical significance in terms of local relapse, distant metastases and overall survival. Interestingly, ALDH1 was associated with favourable prognosis.

Conclusion:

CSC markers are linked with poor radiotherapy outcome in HNSCC, with integrin-β1 being the strongest and independent prognostic factor. Targeting CSC molecules with monoclonal antibodies or pharmaceutical agents may prove important for the treatment of HNSCC.     

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

Background

Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells.

Materials and methods

A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ?-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes.

Results

A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells.

Conclusions

CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.     

Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition

Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/or lost function of Pten. In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.     

Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in human head and neck squamous cell carcinoma

Constitutively activated nuclear factor-kappaB (NF-kappaB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-kappaB in a series of HNSCC cell lines. We confirmed that NF-kappaB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-kappaB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappaB (IkappaBalpha), the natural inhibitor of NF-kappaB. Constitutive NF-kappaB activation in HNSCC was found to be due to constitutive activation of IkappaBalpha kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IkappaBalpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappaB activation. The expression of constitutively active NF-kappaB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappaB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-beta-activated kinase 1 (TAK1), DN-kappa-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-kappaB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappaB and its ligand in HNSCC cells but not interleukin (IL)-1beta. All HNSCC cell lines expressed IL-6, a NF-kappaB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappaB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappaB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.     

Dysregulation of splicing proteins in head and neck squamous cell carcinoma

Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC.     

CIP2A is overexpressed in human ovarian cancer and regulates cell proliferation and apoptosis

CIP2A is a recently characterized oncoprotein which involves in the progression of several human malignancies. This study aimed to investigate its clinical significance and biological function in ovarian cancer. CIP2A expression was analyzed in 152 archived ovarian cancer specimens using immunohistochemistry. One hundred cases (65.79?%) showed CIP2A overexpression, including 63 of 92 serous carcinomas (68.48?%), 21 of 33 endometrioid carcinomas (63.64?%), 12 of 23 mucinous carcinomas (52.17?%), and 4 of 4 clear cell carcinomas (100?%). There is no significant difference of CIP2A expression between serous tumors and all other morphologies combined. CIP2A overexpression positively correlated with advanced FIGO stage (p?=?0.0336) and tumor grade (p?=?0.0213). siRNA knockdown was performed in A2780 and SKOV3 cell lines. MTT, colony formation assay, and flow cytometry were carried out to assess the role of CIP2A in proliferation, cell cycle, and apoptosis. CIP2A depletion in ovarian cancer cell lines inhibited proliferation, blocked cell cycle progression, and increased paclitaxel-induced apoptosis. Futhermore, CIP2A depletion downregulated cyclin D1, c-myc, phospho-Rb, Bcl-2, and phospho-AKT expression. These results validate the role of CIP2A as a clinically relevant oncoprotein and establish CIP2A as a promising therapeutic target of ovarian cancer.     

Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma

Radiotherapy remains the backbone of head and neck cancer therapy but response is sometimes impeded by tumor radioresistance. Identifying predictive biomarkers of radiotherapy response is a crucial step towards personalized therapy. The aim of this study was to explore gene expression data in search of biomarkers predictive of the response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). Microarray analysis was performed on five cell lines with various intrinsic radiosensitivity, selected from a panel of 29 HNSCC cell lines. The bioinformatics approach included Gene Ontology (GO) enrichment profiling and Ingenuity Pathway Analysis (IPA). The GO-analysis detected 16 deregulated categories from which development, receptor activity, and extracellular region represented the largest groups. Fourteen hub genes (CEBPA, CEBPB, CTNNB1, FN1, MYC, MYCN, PLAU, SDC4, SERPINE1, SP1, TAF4B, THBS1, TP53 and VLDLR) were identified from the IPA network analysis. The hub genes in the highest ranked network, (FN1, SERPINE1, THBS1 and VLDLR) were further subjected to qPCR analysis in the complete panel of 29 cell lines. Of these genes, high FN1 expression associated to high intrinsic radiosensitivity (p=0.047). In conclusion, gene ontologies and hub genes of importance for intrinsic radiosensitivity were defined. The overall results suggest that FN1 should be explored as a potential novel biomarker for radioresistance.     

CIP2A-mediated Akt activation plays a role in bortezomib-induced apoptosis in head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) is a worldwide disease with aggressive course and dismal outcome. Bortezomib, a proteasome inhibitor, has been approved clinically for hematological malignancies and demonstrated to have activities against solid tumors in vitro through inhibition of NF-kB activity. Here, we disclose that bortezomib induced apoptosis of HNSCC cells in vitro and in vivo through inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A)-mediated PP2A dependent Akt activation. HNSCC cells, including Ca9-22, SAS, and SCC-25, were treated with bortezomib and evaluated for viability, apoptosis, and signal transduction. Three HNSCC cells, including Ca9-22, SAS, and SCC-25, were sensitive to bortezomib with marked growth inhibition and apoptosis. We found phospho-Akt (p-Akt, Ser473) played a significant role in bortezomib-induced apoptosis. The activity of PP2A was significantly increased after the treatment of bortezomib without alternation of PP2A level or the dynamic interaction of PP2A-Akt. Silencing PP2A by small interference RNA (siRNA) abolished bortezomib-induced Akt inhibition and apoptosis. In addition, bortezomib inhibited CIP2A in pre-translational level in a dose- and time-dependent manner. Over-expression of CIP2A up-regulated p-Akt and protected HNSCC cells from bortezomib-induced apoptosis. Furthermore, xenograft model showed that bortezomib down-regulated CIP2A and p-Akt in SAS tumor cells. CIP2A is demonstrated to be a new therapeutic target of bortezomib in HNSCC.     

The Akt inhibitor KP372-1 inhibits proliferation and induces apoptosis and anoikis in squamous cell carcinoma of the head and neck

Therapies that target signaling pathways critical to the pathogenesis and progression of squamous cell carcinoma of the head and neck (HNSCC) are needed. One such target, phosphatidylinositol 3-kinase, and its downstream target serine/threonine kinase, Akt, are up-regulated in HNSCC. Targeted therapy could consist of inhibitors of these kinases or, alternatively, of inhibitors of the pathways that they regulate. To explore the effect of Akt inhibition on the growth and survival of HNSCC tumors, we evaluated the effect of a novel Akt inhibitor, KP372-1, on the growth, survival, and sensitivity to anoikis of HNSCC cell lines in culture. Using Western blotting of head and neck cancer cell lines and squamous mucosa and carcinoma specimens, we found that Akt was highly phosphorylated in head and neck cancer cell lines and human head and neck squamous carcinoma specimens. Treatment of HNSCC cell lines with KP372-1 blocked the activation of Akt, inhibited head and neck cancer cell proliferation, and induced apoptosis and anoikis in several HNSCC cell lines. Furthermore, KP372-1 decreased the phosphorylation of the S6 ribosomal (Ser240/244) protein, which is a downstream target of Akt. Taken together, these findings indicate that KP372-1 may be a useful therapeutic agent for HNSCC and should be further evaluated in preclinical models of HNSCC.     

BMSC enhance the survival of paclitaxel treated squamous cell carcinoma cells in vitro

The 5-year survival rate of patients suffering from head and neck squamous cell carcinoma (HNSCC) is unsatisfying despite the advances in carcinoma treatment. Recent studies suggest that stem cells can be used as a gene therapy carrier for cancer treatment. Stem cells produce different cytokines such as growth factors in a paracrine manner and cancer cells may show drug resistance in the presence of such growth factors. Reports in the literature concerning treatment of cancer using bone marrow derived stem cells (BMSC) are controversial, which led us to investigate the effects of paclitaxel on human HNSCC cell lines (FaDu and HLaC 78) cultivated simultaneously with BMSC in a transwell system (co-culture). Co-culture and HNSCC cell lines were treated with 10nM of paclitaxel for 24h. Morphology, viability and apoptosis were measured by microscopy, the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the Annexin V-propidium iodide test. The survival of HNSCC cell lines treated with paclitaxel in co-culture increased significantly compared to control cells. Apoptosis of HNSCC cell lines in co-culture was attenuated significantly. In conclusion, BMSC increase HNSCC resistance to treatment with paclitaxel in vitro. Tumor-stroma interactions are critical components of tumor biology including tumor invasion and metastatic potential. Therefore particular attention must be paid to the complex tumor-stroma interactions to fully understand how tumor cells become chemoresistant.