A Novel Role of Connective Tissue Growth Factor in the Regulation of the Epithelial Phenotype.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells lose their adhesive properties and gain invasive, metastatic, and mesenchymal properties. Maintaining the balance between the epithelial and mesenchymal stage is essential for tissue homeostasis. Many of the genes promoting mesenchymal transformation have been identified; however, our understanding of the genes responsible for maintaining the epithelial phenotype is limited. Our objective was to identify the genes responsible for maintaining the epithelial phenotype and inhibiting EMT. METHODS: RNA seq was performed using an vitro model of EMT. CTGF expression was determined via qPCR and Western blot analysis. The knockout of CTGF was completed using the CTGF sgRNA CRISPR/CAS9. The tumorigenic potential was determined using NCG mice. RESULTS: The knockout of CTGF in epithelial ovarian cancer cells leads to the acquisition of functional characteristics associated with the mesenchymal phenotype such as anoikis resistance, cytoskeleton remodeling, increased cell stiffness, and the acquisition of invasion and tumorigenic capacity. CONCLUSIONS: We identified CTGF is an important regulator of the epithelial phenotype, and its loss is associated with the early cellular modifications required for EMT. We describe a novel role for CTGF, regulating cytoskeleton and the extracellular matrix interactions necessary for the conservation of epithelial structure and function. These findings provide a new window into understanding the early stages of mesenchymal transformation.

Dacomitinib and gedatolisib in combination with fractionated radiation in head and neck cancer.

BACKGROUND AND PURPOSE: There has been little success targeting individual genes in combination with radiation in head and neck cancer. In this study we investigated whether targeting two key pathways simultaneously might be more effective. MATERIALS AND METHODS: We studied the effect of combining dacomitinib (pan-HER, irreversible inhibitor) and gedatolisib (dual PI3K/MTOR inhibitor) with radiation in well characterized, low passage xenograft models of HNSCC in vitro and in vivo. RESULTS: Dacomitinib showed differential growth inhibition in vitro that correlated to EGFR expression whilst gedatolisib was effective in both cell lines. Neither agent radiosensitized the cell lines in vitro. In vivo studies demonstrated that dacomitinib was an effective agent alone and in combination with radiation whilst the addition of gedatolisib did not enhance the effect of these two modalities despite inhibiting phosphorylation of key genes in the PI3K/MTOR pathway. CONCLUSIONS: Our results showed that combining two drugs with radiation provided no added benefit compared to the single most active drug. Dacomitinib deserves more investigation as a radiation sensitizing agent in HNSCC.

Dual blockade of PI3K and MEK in combination with radiation in head and neck cancer.

BACKGROUND AND PURPOSE: In this study we have combined fractionated radiation treatment (RT) with two molecular targeted agents active against key deregulated signaling pathways in head and neck cancer. MATERIALS AND METHODS: We used two molecularly characterized, low passage HNSCC cell lines of differing biological characteristics to study the effects of binimetinib and buparlisib in combination with radiation in vitro and in vivo. RESULTS: Buparlisib was active against both cell lines in vitro whereas binimetinib was more toxic to UT-SCC-14. Neither agent modified radiation sensitivity in vitro. Buparlisib significantly inhibited growth of UT-SSC-15 alone or in combination with RT but was ineffective in UT-SCC-14. Binimetinib did cause a significant delay with RT in UT-SCC-14 and it significantly reduced growth of the UT-SCC-15 tumors both alone and with RT. The tri-modality treatment was not as effective as RT with a single effective agent. CONCLUSIONS: No significant benefit was gained by the combined use of the two agents with RT even though each was efficacious when used alone.

Antitumor activity of the dual PI3K/MTOR inhibitor, PF-04691502, in combination with radiation in head and neck cancer.

BACKGROUND AND PURPOSE: Head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge where new treatments are required to supplement the current-standard-of care of concurrent chemoradiation. The PI3K/AKT/MTOR pathway has been identified from several next generation DNA sequencing studies to be commonly altered and activated in HNSCC. MATERIAL AND METHODS: In this study we investigated the activity of PF-04691502, an orally active ATP-competitive, dual inhibitor of PI3K and mTOR, in combination with a clinically relevant fractionated radiation treatment in two contrasting, well characterized, low passage HNSCC models. RESULTS: We found that PF-04691502 combined synergistically with radiation in the UT-SCC-14 model derived from a primary cancer but was ineffective in the UT-SCC-15 model which was derived from a nodal recurrence. Further examination of the status of key signaling pathways combined with next generation DNA sequencing of a panel of 160 cancer-associated genes revealed crucial differences between the two models that could account for the differential effect. The UT-SCC-15 cell line was characterized by a higher mutational burden, an excess of variants in the PI3K/AKT/MTOR pathway, increased constitutive activity of PI3K, AKT1 and 2 and MTOR and an inability to inhibit key phosphorylation events in response to the treatments. CONCLUSION: This study clearly highlights the promise of agents such as PF-04691502 in selected HNSCCs but also emphasizes the need for molecular characterization and alternative treatment strategies in non-responsive HNSCCs.

The significance of Trk receptors in pancreatic cancer.

This study investigated the Trk receptor family as a therapeutic target in pancreatic ductal adenocarcinoma and assessed their prognostic significance. Global gene expression analysis was investigated in prospectively collected pancreatic ductal adenocarcinomas that had either undergone neoadjuvant chemoradiation or were treated by surgery. PANC-1 and MIA-PaCa-2 cell lines were investigated to establish whether fractionated radiation altered expression of four neuroendocrine genes and whether this resulted in subsequent changes in radiosensitivity. A specific inhibitor of TrkA, B, and C, AstraZeneca 1332, was investigated in vitro and in vivo in combination with radiation. A tissue microarray was constructed from 77 pancreatic ductal adenocarcinoma patients who had undergone neoadjuvant chemoradiation and the Trk receptor, and neurogenic differentiation 1 expression was assessed and correlated with overall survival. A total of 99 genes were identified that were differentially expressed in the chemoradiation patients with neuroendocrine genes and pathways, in particular the neurogenic differentiation 1 and Trk receptor family, being prominent. Fractionated radiation upregulated the expression of neuroendocrine genes, and AstraZeneca 1332 treatment in vitro enhanced radiosensitivity. No added effect of AstraZeneca 1332 was observed in vivo. Trk receptor expression varied between isoforms but did not correlate significantly with clinical outcome. Radiation treatment upregulated neuroendocrine gene expression but the Trk receptor family does not appear to be a promising treatment target.

Cancer Stem Cell Signaling during Repopulation in Head and Neck Cancer.

The aim of the study was to investigate cancer stem signaling during the repopulation response of a head and neck squamous cell cancer (HNSCC) xenograft after radiation treatment. Xenografts were generated from low passage HNSCC cells and were treated with either sham radiation or 15 Gy in one fraction. At different time points, days 0, 3, and 10 for controls and days 4, 7, 12, and 21, after irradiation, 3 tumors per group were harvested for global gene expression, pathway analysis, and immunohistochemical evaluation. 316 genes were identified that were associated with a series of stem cell-related genes and were differentially expressed (p ≤ 0.01 and 1.5-fold) at a minimum of one time point in UT-SCC-14 xenografts after radiation. The largest network of genes that showed significant changes after irradiation was associated with CD44, NOTCH1, and MET. c-MET and ALDH1A3 staining correlated with the changes in gene expression. A clear pattern emerged that was consistent with the growth inhibition data in that genes associated with stem cell pathways were most active at day 7 and day 12 after irradiation. The MET/CD44 axis seemed to be an important component of the repopulation response.

Crizotinib Fails to Enhance the Effect of Radiation in Head and Neck Squamous Cell Carcinoma Xenografts.

AIM: Mesenchymal-epithelial transition factor (MET), a receptor tyrosine kinase, is expressed in head and neck squamous cell carcinomas (HNSCC) and is involved in tumor progression and associated with poor prognosis. MET can be inhibited by crizotinib, a potent ATP-competitive kinase inhibitor. We examined the effects of combining crizotinib and radiation in a pre-clinical HNSCC model. MATERIALS AND METHODS: Nine HNSCC cell lines were screened for MET expression, copy-number amplification and mutational status. The in vitro effects of crizotinib and radiation were assessed with clonogenic survival assays. MET signaling proteins were assessed with western blot and receptor tyrosine kinase array. Tumor growth-delay experiments with UT-SCC-14 and UT-SCC-15 oral tongue xenografts were used to assess in vivo tumor radiosensitivity. RESULTS: All nine HNSCC cell lines showed a varying degree of MET protein and RNA expression. Increased MET copy number was not present. MET was expressed after irradiation both in vitro and in vivo. Crizotinib alone inhibited phosphorylation of MET and inhibited cell growth in vitro but did not inhibit phosphorylation of downstream signaling proteins: MAPK, AKT or c-SRC. When combined with radiation in vitro, crizotinib demonstrated radiation enhancement in only one cell line. Crizotinib did not enhance the effect of radiation in either UT-SCC-14 or UT-SCC-15 tumors grown as xenografts. CONCLUSION: MET is overexpressed in HNSCC cell lines, however, crizotinib failed to enhance the radiation response and failed to inhibit MET downstream signaling proteins in this HNSCC model.

The Effects of Pulsed Radiation Therapy on Tumor Oxygenation in 2 Murine Models of Head and Neck Squamous Cell Carcinoma.

PURPOSE: To evaluate the efficacy of low-dose pulsed radiation therapy (PRT) in 2 head and neck squamous cell carcinoma (HNSCC) xenografts and to investigate the mechanism of action of PRT compared with standard radiation therapy (SRT). METHODS AND MATERIALS: Subcutaneous radiosensitive UT-SCC-14 and radioresistant UT-SCC-15 xenografts were established in athymic NIH III HO female mice. Tumors were irradiated with 2 Gy/day by continuous standard delivery (SRT: 2 Gy) or discontinuous low-dose pulsed delivery (PRT: 0.2 Gy × 10 with 3-min pulse interval) to total doses of 20 Gy (UT14) or 40 Gy (UT15) using a clinical 5-day on/2-day off schedule. Treatment response was assessed by changes in tumor volume, (18)F-fluorodeoxyglucose (FDG) (tumor metabolism), and (18)F-fluoromisonidazole (FMISO) (hypoxia) positron emission tomography (PET) imaging before, at midpoint, and after treatment. Tumor hypoxia using pimonidazole staining and vascular density (CD34 staining) were assessed by quantitative histopathology. RESULTS: UT15 and UT14 tumors responded similarly in terms of growth delay to either SRT or PRT. When compared with UT14 tumors, UT15 tumors demonstrated significantly lower uptake of FDG at all time points after irradiation. UT14 tumors demonstrated higher levels of tumor hypoxia after SRT when compared with PRT as measured by (18)F-FMISO PET. By contrast, no differences were seen in (18)F-FMISO PET imaging between SRT and PRT for UT15 tumors. Histologic analysis of pimonidazole staining mimicked the (18)F-FMISO PET imaging data, showing an increase in hypoxia in SRT-treated UT14 tumors but not PRT-treated tumors. CONCLUSIONS: Differences in (18)F-FMISO uptake for UT14 tumors after radiation therapy between PRT and SRT were measurable despite the similar tumor growth delay responses. In UT15 tumors, both SRT and PRT were equally effective at reducing tumor hypoxia to a significant level as measured by (18)F-FMISO and pimonidazole.

Glucose metabolism gene expression patterns and tumor uptake of ¹8F-fluorodeoxyglucose after radiation treatment.

PURPOSE: To investigate whether radiation treatment influences the expression of glucose metabolism genes and compromises the potential use of (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) as a tool to monitor the early response of head and neck cancer xenografts to radiation therapy (RT). METHODS AND MATERIALS: Low passage head and neck squamous cancer cells (UT14) were injected to the flanks of female nu/nu mice to generate xenografts. After tumors reached a size of 500 mm(3) they were treated with either sham RT or 15 Gy in 1 fraction. At different time points, days 3, 9, and 16 for controls and days 4, 7, 12, 21, 30, and 40 after irradiation, 2 to 3 mice were assessed with dynamic FDG-PET acquisition over 2 hours. Immediately after the FDG-PET the tumors were harvested for global gene expression analysis and immunohistochemical evaluation of GLUT1 and HK2. Different analytic parameters were used to process the dynamic PET data. RESULTS: Radiation had no effect on key genes involved in FDG uptake and metabolism but did alter other genes in the HIF1α and glucose transport-related pathways. In contrast to the lack of effect on gene expression, changes in the protein expression patterns of the key genes GLUT1/SLC2A1 and HK2 were observed after radiation treatment. The changes in GLUT1 protein expression showed some correlation with dynamic FDG-PET parameters, such as the kinetic index. CONCLUSION: (18)F-fluorodeoxyglucose positron emission tomography changes after RT would seem to represent an altered metabolic state and not a direct effect on the key genes regulating FDG uptake and metabolism.

Gene expression changes during repopulation in a head and neck cancer xenograft.

BACKGROUND/PURPOSE: To investigate temporal changes in global gene expression and pathways involved in the response to irradiation during phases of growth inhibition, recovery and repopulation in a human head and neck squamous cell cancer (HNSCC) xenograft. METHODS AND MATERIALS: Low passage head and neck squamous cancer cells (UT-14-SCC) were injected into the flanks of female nu/nu mice to generate xenografts. After tumors reached a size of 500 mm3, they were treated with either sham RT or 15Gy in one fraction. At different time points, days 0, 3, and 10 for controls and days 4, 7, 12, and 21 after irradiation, the tumors were harvested for global gene expression analysis and pathway analysis. RESULTS: The tumors showed growth inhibition through days 4-7 and began the transition to regrowth around the day 12 time point. When comparing the pooled controls to each day of treatment, there were 22, 119, 125, and 25 differentially expressed genes on days 4, 7, 12, and 21 respectively using a p⩽0.01 and a 2-fold cut-off. Gene Ontology (GO), gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) identified different biological processes, cell process pathways and expression targets to be active on each time point after irradiation. An important observation was that the molecular events on day 12 which represented the transition from growth inhibition to regrowth identified interferon and cytokine related genes and signaling pathways as the most prominent. CONCLUSION: The findings in this study compliment research which has identified components of interferon-related signaling pathways to be involved in radioresistance. Further work will be required to understand the significance of these genes in both radioresistance and treatment response leading to new therapeutic strategies and prognostic tools.

Isolation and genomic characterization of stem cells in head and neck cancer.

BACKGROUND: This study investigated the use of 3 different established cell-sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines. METHODS: Five low-passage cell lines were subjected to cell sorting based on Hoechst side population, Aldefluor, and CD44 expression. Isolated cell populations were studied for gene expression, radiosensitivity, and chemosensitivity to cisplatin and paclitaxel. RESULTS: Each sorting method identified a different set of genes associated with different gene ontology categories, with mitosis being the only common category. CD44-associated gene changes were almost exclusively associated with cell cycle and in particular mitosis. There were no significant differences in radiosensitivity or cisplatin sensitivity of stem or non-stem cells, but CD44-isolated stem cells were more resistant to paclitaxel. CONCLUSIONS: This study suggested that CD44 may be the most promising cell-sorting strategy to isolate and investigate the impact of stem cells in head and neck squamous cell cancer (HNSCC).

TMPRSS2/ERG fusion gene expression alters chemo- and radio-responsiveness in cell culture models of androgen independent prostate cancer.

PURPOSE/OBJECTIVES: The androgen regulated transmembrane serine protease (TMPRSS2) and ETS transcription factor (ERG) gene fusion is a strong prognostic factor for disease recurrence following prostatectomy. Expression of TMPRSS2/ETS-related gene (ERG) fusion gene transcripts is linked with tumor proliferation, invasion, and an aggressive phenotype. The aim of this study was to define the effect of TMPRSS2/ERG fusion gene expression on chemo- and radiosensitivity in prostate tumor cell lines. MATERIALS/METHODS: Clonogenic survival of PC3 and DU145 cells stably expressing TMPRSS2/ERG Types III and VI fusion genes was measured after X-irradiation (0-8 Gy) and Paclitaxel. Cell cycle changes and DNA double-strand break induction and repair were assessed. Differential gene expression was measured by microarray analysis. ERG signaling pathway interactions were studied using Ariadne Pathway Studio. RESULTS: Expression of the TMPRSS2/ERG fusions in PC3 cells increased radiation sensitivity and decreased paclitaxel sensitivity. Increased radiosensitivity was associated with persistent DNA breaks 24 hr post-irradiation, down-regulation of genes involved in DNA repair and mitosis and up-regulation of ETV, an ETS transcription factor. However, DU145 Types III and VI demonstrated a different sensitivity phenotype and gene expression changes. Pathway analysis of ERG signaling further illustrated the variation between the PC3 and DU145 cell lines containing TMPRSS2/ERG fusions. CONCLUSIONS: The effect of TMPRSS2/ERG gene fusions had differing effects on radiosensitivity and chemosensitivity depending on cell line and fusion type. Further work is needed with clinical samples to establish whether TMPRSS2/ERG gene fusions affect radio- and chemosensitivity in vivo.

Sorafenib and radiation: a promising combination in colorectal cancer.

PURPOSE: To examine the combination of radiation and the multikinase inhibitor sorafenib in human colorectal cancer cell lines and xenografts. METHODS AND MATERIALS: HT29 and SW48 colorectal cancer cells were studied in vitro using MTT assays to establish the optimal timing of radiation and sorafenib. This optimal timing was then investigated in clonogenic survival assays. Xenografts were established, and the effect of a 3-week schedule of daily radiation and sorafenib was studied by growth delay. RESULTS: Sorafenib predominantly had minimal effects on cell growth or radiation response in MTT growth assays, though growth inhibition was significantly enhanced in HT29 cells when sorafenib was administered after radiation. The highest dose of sorafenib altered the alpha component of the cell survival curve in clonogenic assays. The combination of radiation and sorafenib was synergistic in SW48 xenografts, with a mean time to threshold tumor size of 11.4 +/- 1.0 days, 37.0 +/- 9.5 days, 15.5 +/- 3.2 days, and 98.0 +/- 11.7 days in the control, radiation, sorafenib, and combined treatment group, respectively. The effect on HT29 tumors was additive, with mean time to threshold volume of 12.6 +/- 1.1 days, 61.0 +/- 4.3 days, 42.6 +/- 11.7 days, and 100.2 +/- 12.4 days. CONCLUSIONS: Sorafenib had little effect on radiation response in vitro but was highly effective when combined with radiation in vivo, suggesting that inhibition of proliferation and interference with angiogenesis may be the basis for the interaction.