Squamous Cell Differentiation Research Articles

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells

BackgroundEpidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells.MethodsImmortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated.ResultsCells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR inhibitors in epithelial-like cells. Furthermore, mesenchymal T-Epi cells showed resistance to EGFR inhibitors by circumventing the dephosphorylation of EGFR signaling. Cetuximab consistently showed antitumor effects, and increased involucrin expression in TE-11R (epithelial-like)-derived xenograft tumors but not TE-8 (mesenchymal-like)-derived xenograft tumors.ConclusionsThe factor determining the therapeutic effects of EGFR inhibitors in ESCC cells is the phenotype representing the epithelial-like or mesenchymal-like cells. Mesenchymal-like ESCC cells are resistant to EGFR inhibitors because EGFR signaling is not blocked. EGFR inhibitors show antitumor effects on epithelial-like ESCC cells accompanied by promotion of squamous cell differentiation.

Undifferentiated carcinoma of the mesentery in a 51 year old male: A Case Report

Undifferentiated carcinoma is a usually aggressive, malignant epithelial neoplasm composed of atypical cells which do not display evidence of glandular, squamous, or urothelial cell differentiation.1 Mesenteric tumors are rare and consist of a heterogeneous group of lesions. Among them undifferentiated carcinoma of the mesentery is very very rare entity. Here we present a case of undifferentiated carcinoma of the mesentery in a 51 year old male admitted to the department of surgery, Shaheed Suhrawardy Medical College Hospital (ShSMCH) with rapidly increasing huge abdominal lump, central abdominal pain and weight loss. Ultrasound of the whole abdomen and CT scan of whole abdomen reports were suggestive of retroperitoneal mass. Exploratory laparotomy was done and specimen sent for histopathology. Reports were suggestive of malignant mesothelioma whereas immunohistochemistry report revealed undifferentiated carcinoma of the mesentery. To the best of our knowledge, there was no report of undifferentiated carcinoma of the mesentery in a 51 year old male from Bangladesh or any other countryJ Shaheed Suhrawardy Med Coll, December 2015, Vol.7(2); 79-81

Vimentin regulates differentiation switch via modulation of keratin 14 levels and their expression together correlates with poor prognosis in oral cancer patients.

Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchymal origin, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In view of the perceived role of keratins (Ks) as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through the reprogramming of keratins, in transformed cells. To address this, vimentin was stably downregulated in oral cancer derived cells. Further, global keratin profiling was performed after high salt keratin extraction. K5/K14 pair was found to be significantly downregulated, both at protein and mRNA levels upon vimentin downregulation. The previous study from our laboratory has shown a role of the K5/K14 pair in proliferation and differentiation of squamous epithelial cells. Vimentin depleted cells showed an increase in the differentiation state, marked by an increase in the levels of differentiation specific markers K1, involucrin, filaggrin and loricrin while its proliferation status remained unchanged. Rescue experiments with the K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased differentiation state. ΔNp63 emerged as one of the indirect targets of vimentin, through which it modulates the expression levels of K5/K14. Further, immunohistochemistry showed a significant correlation between high vimentin-K14 expression and recurrence/poor survival in oral cancer patients. Thus, in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression. Moreover, vimentin-K14 together may prove to be the novel markers for the prognostication of human oral cancer.

Detection and oncological effect of circulating tumour cells in patients with variant urothelial carcinoma histology treated with radical cystectomy.

To investigate for the presence of circulating tumour cells (CTC) in patients with variant urothelial carcinoma of the bladder (UCB) histology treated with radical cystectomy (RC), and to determine their impact on oncological outcomes. We prospectively collected data of 188 patients with UCB treated with RC without neoadjuvant chemotherapy. Pathological specimens were meticulously reviewed for pure and variant UCB histology. Preoperatively collected blood samples (7.5 mL) were analysed for CTC using the CellSearch® system (Janssen, Raritan, NJ, USA). Variant UCB histology was found in 47 patients (25.0%), most frequently of squamous cell differentiation (16.5%). CTC were present in 30 patients (21.3%) and 12 patients (25.5%) with pure and variant UCB histology, respectively. At a median follow-up of 25 months, the presence of CTC and non-squamous cell differentiation were associated with reduced recurrence-free survival (RFS) and cancer-specific survival (pairwise P ≤ 0.016). Patients without CTC had better RFS, independent of UCB histology, than patients with CTC with any UCB histology (pairwise P < 0.05). In multivariable analyses, the presence of CTC, but not variant UCB histology, was an independent predictor for disease recurrence [hazard ratio (HR) 3.45; P < 0.001] and cancer-specific mortality (HR 2.62; P = 0.002). CTC are detectable in about a quarter of patients with pure or variant UCB histology before RC, and represent an independent predictor for outcomes, when adjusting for histological subtype. In addition, our prospective data confirm the unfavourable influence of non-squamous cell-differentiated UCB on outcomes.

P40 &amp; thyroid transcription factor-1 immunohistochemistry: A useful panel to characterize non-small cell lung carcinoma-not otherwise specified (NSCLC-NOS) category

Background & objectives:Accurate histopathological subtyping of non-small cell lung carcinoma (NSCLC) is essential for targeted therapeutic agents. Immunohistochemistry (IHC) is helpful in identification of different tumour subtypes. In this study two marker approaches, one each for glandular and squamous cell differentiation was applied to maximize the proportion of accurately subtyped NSCLC not otherwise specified (NOS) tumours on small biopsy samples.Methods:Two hundred and sixty three consecutive lung biopsies of primary lung carcinoma were prospectively studied. These were subtyped first morphologically and then by IHC for p40 and thyroid transcription factor-1 (TTF-1). The diagnosis of NSCLC-NOS before and after addition of IHC was evaluated. Results were correlated and validated with morphologically proven cases and matched surgical specimens.Results:Based on morphology, only 140 of the 263 (53.2%) cases of NSCLC were characterized, whereas 123 (46.7%) were classified as NSCLC-NOS type. With addition of IHC (p40 and TTF-1), the latter category reduced to 14.4 per cent and a sum of 225 (85.5%) cases were accurately subtyped into squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma. p40 showed 100 per cent sensitivity and specificity for squamous differentiation whereas TTF-1 showed sensitivity of 85.3 per cent and specificity of 98.1 per cent. Ninety per cent correlation of morphologic subtypes was achieved with matched resected specimens.Interpretation & conclusions:Our results showed that an approach of using only a two-antibody panel (p40 and TTF-1) might help in reduction of diagnostic category of NSCLC-NOS significantly and contribute in saving tissue for future molecular testing.

Abstract 1168: The codon 389 polymorphism of PICT-1 is a risk factor for human cervical cancer

Abstract The uterine cervical cancers profoundly involve in the infection with human papillomavirus (HPV), and cause the deaths of more than 250,000 women all over the world. To date, the carcinogenesis of uterine cervical cancers was not fully understood, then we have to investigate more precise genetic and molecular events to develop early diagnosis and identify new therapeutic targets. The PICT-1 (Protein Interacting with Carboxyl Terminus-1), one of the nucleolar proteins, has tumor suppressor functions, but its association with uterine cervical carcinogenesis has been unknown. So we investigate PICT-1 gene mutations, polymorphism on codon 389 (rs184994), and protein expression levels to investigate the association with pathogenesis of uterine cervical cancers. Firstly we found missense mutations of functionally important regions of PICT-1 in one of seven uterine cervical cancer cell lines (14%) and one of 10 surgical samples (10%). Next we focused on codon 389 polymorphism (Gln to Arg) which located in important regions of PICT-1 function. In result there was a statistically significant association between PICT-1 codon 389 polymorphism and the risk of uterine cervical cancers: A/G (odds ratio 6.48, 95% confidence interval 1.98-21.22, P = 0.0012), G/G (odds ratio 3.15, 95% confidence interval 1.03-9.61, P = 0.0403), and A/G or G/G (odds ratio 4.44, 95% confidence interval 1.62-12.23, P = 0.0026). We also identified PICT-1 protein level was reduced in uterine cervical cancer cell lines by Western blotting and cervical cancer tissues by immunohistochemistry staining (IHC). In IHC analysis, expression of PICT-1 in adjacent normal cervix was found in nucleus, especially nucleolus, but in differentiating cells layers located upper parabasal cell layer, PICT-1 also expressed in cytoplasm and gradually reduced the expression of nucleolus. In patient tissues, compared to normal epithelium, we found reduction of PICT-1 expression in most samples found even in uterine cervical cancers at early invasive lesions, and weaker PICT-1 expression was observed in more invasive lesions. Interestingly, we also detected high expressed and/or re-expressed PICT-1 in cytoplasm and/or nucleolus of keratinized lesions of cervical cancers. Our results indicate that disruption of PICT-1 by gene mutation and/or reduction of protein level may associate with the pathogenesis of uterine cervical cancers, and its codon 389 polymorphism may increase the risk of uterine cervical cancers. And also we suggested that PICT-1 located in cytoplasm may be important to squamous cell differentiation, and we speculated the further study of association between PICT-1 and cell differentiation would be of value to the field of the differentiation-inducing therapy for many hard-to-treat patients of many cancers, including uterine cervical cancers. Citation Format: Masafumi Yoshimoto, Aoi Tokuda, Shoko Takahashi, Yuji Yaginuma. The codon 389 polymorphism of PICT-1 is a risk factor for human cervical cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1168.

MP38-02 THE AMOUNT OF INTRA- AND POSTOPERATIVE BLOOD TRANSFUSION IN BLADDER CANCER PATIENTS WITH RADICAL CYSTECTOMY IS AN INDEPENDENT PROGNOSTIC FACTOR FOR POOR OUTCOME – RESULTS OF A SINGLE-CENTER STUDY

INTRODUCTION AND OBJECTIVES: Extra nodal extension (ENE) is associated with poor outcomes in lymph node (LN) positive bladder cancer patients treated with radical cystectomy (RC). In addition, variant histologiesofurothelial carcinomaof thebladder (UCB)seemtobeassociated with unfavorable outcomes after RC. The impact of variant UCB histologies on ENE, however, remains unclear. To investigate the association of variant UCB histologies and ENE as well as their predictive value on outcomes. METHODS: We collected data of 517 UCB patients treated with RC without neoadjuvant chemotherapy at our academic center between 1996 and 2011. All pathologic bladder and LN specimen were meticulously reviewed for presence and extent of variant UCB histologies as well as presence and extent of ENE by dedicated uro-pathologists. For analyses we used the following subgroups: Pure UCB vs. UCB and squamous cell carcinoma vs. UCB and non-squamous cell carcinoma, respectively. Cox regression models evaluated the association with disease recurrence and cancer-specific survival, respectively. RESULTS: Overall, 138 patients had LN metastasis (27%). The median number of removed LNs was 15 (IQR 9; 18). 43 LN positive patients (31%) had ENE with a median length of 1cm. UCB with variant histology was present in 96 patients (20%) with squamous cell (12.4%) and sarcomatoid (2.7%) differentiation beingmost common. The general presence of variant histology and any variant histology subtype were neither associated with lymph node metastasis nor ENE (all p-values1⁄4n.s.). Compared to pure UCB, variant histology of non-squamous cell differentiation, but not squamous cell differentiation, was associated with disease recurrence and cancer-specific mortality, respectively (pairwise p-values 1⁄40.016) in Kaplan-Meier analyses. In LN positive patients, the presence and any subgroup did not influence outcomes (p1⁄4n.s.). The presence of LNmetastasis and ENE in LN positive patients were significantly associated with disease recurrence and cancer-specific mortality, respectively (all p 1cm length was also associated with disease recurrence (p1⁄40.049). In multivariable analyses that adjusted for standard UCB prognosticators, ENE, but not variant histology,was an independent predictor for disease recurrence (HR1.55; p1⁄40.016) and cancer-specific mortality (HR 1.50; p1⁄40.008). CONCLUSIONS: ENE is a strong predictor for disease recurrence and cancer-specific mortality in node positive pure UCB and UCB with variant histology. Presence of variant histology does not seem to increase the likelihood of ENE.

Inveterate squamous cell carcinoma of the upper eyelid: a case report

Squamous cell carcinoma (SCC) is a malignant tumor of epithelium that shows squamous cell differentiation. It is the second most common cancer of the skin and usually occurs in areas exposed to the sun but it can rarely arise within the conjunctival epithelium with a deep component. We describe a woman with a history of chronic blepharoconjunctivitis unresponsive to topical medications. Examination disclosed a hyperaemic translucent patch with blurred margins of the upper palpebral conjunctiva. Tarsoconjunctival biopsy revealed intraepithelial squamous cell carcinoma. Management consisted of complete tumor excision with removal of the entire posterior lamella of the left upper eyelid and reconstruction. Histopathologic analysis confirmed primary squamous cell carcinoma arising from conjunctival epithelium, involving the underlying tarsus. Patients with unexplained chronic unilateral blepharoconjunctivitis or papillary hypertrophy of the palpebral conjunctiva should be considered for biopsy to rule out neoplasia, even when there is no sign of an evident mass.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC.

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongly suggest that downregulation of S100A8/A9 through epigenetic mechanisms may contribute to increased proliferation, malignant transformation, and disease progression in HNSCC.

Specific types of bladder cancer

Bladder cancer shows rare variants and special subtypes with diverse prognostic importance and therefore may necessitate different therapeutic approaches. For pathologists it is important to histologically diagnose and specify such variants. Nested variants of urothelial carcinoma with inconspicuous, well-formed tumor cell nests present with an aggressive course. The plasmacytoid variant, which morphologically resembles plasma cells is associated with a shorter survival time and a high frequency of peritoneal metastasis. Micropapillary urothelial carcinoma with small papillary tumor cell islands within artificial tissue retraction spaces and frequent lymphovascular invasion also has a poor prognosis. Other important rare differential variants listed in the World Health Organization (WHO) classification are microcystic, lymphoepithelioma-like, sarcomatoid, giant cell and undifferentiated urothelial carcinomas. Additionally, there are three special types of bladder cancer: squamous cell carcinoma, adenocarcinoma and small cell neuroendocrine carcinoma of the bladder. These tumors are characterized by pure squamous cell or glandular differentiation and are sometimes less responsive to adjuvant (chemo)therapy. Small cell carcinoma of the bladder mimics the neuroendocrine features of its pulmonary counterpart, shows an aggressive course but is sensitive to (neo-)adjuvant chemotherapy. The morphology and histology of the most important variants and special types are discussed in this review.

Prognostic potential of AgNORs in oral submucous fibrosis

Aim and Objective:The role of prognosis cannot be stressed enough, especially when it comes to potentially malignant lesions. The argyrophilic nucleolar organizer regions (AgNORs), which is simple and cost-effective has been used in diagnostic and prognostic pathologies. This study seeks to identify the nucleolar organizer regions (NORs) in oral submucous fibrosis (OSMF), to correlate the AgNOR count with the histologic grade of OSMF, and to evaluate the prognostic potential of AgNOR.Materials and Methods:The sample size consisted of archival paraffin blocks of 35 cases of varying grades of OSMF and 10 cases of squamous cell carcinoma. Normal mucosa samples served as controls for the study. AgNOR staining in accordance with the method of Smith and Crocker was performed and Student's t-test was used for statistical analysis.Results:The results showed an increase in AgNOR counts with corresponding grades of OSMF, the count being least in normal mucosa and also an increase in AgNOR count with corresponding decrease in differentiation of oral squamous cell carcinoma.Conclusion:AgNOR staining is a rapid and inexpensive procedure representing cellular proliferation that can be used to assess the nature of the lesion and therefore, the prognosis.

Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10.

Our previous reports demonstrated that the magnitude of natural killer (NK) cell-mediated cytotoxicity correlate directly with the stage and level of differentiation of tumor cells. In addition, we have shown previously that activated NK cells inhibit growth of cancer cells through induction of differentiation, resulting in the resistance of tumor cells to NK cell-mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report, we show that in comparison to IL-2 + anti-CD16mAb-treated NK cells, activation of NK cells by probiotic bacteria (sAJ2) in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of oral squamous cancer stem cells, MIA PaCa-2 stem-like/poorly differentiated pancreatic tumors, and healthy stem cells of apical papillae through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell-mediated killing induced by IL-2 + anti-CD16mAb + sAJ2-treated NK cells is induced by combination of IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, were able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC-I on NK-differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergize with NK cells in the presence of probiotic bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell-mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, and decreased inflammatory cytokine release.

Histomolecular profiling of pleomorphic, spindle cell, and giant cell carcinoma of the lung for targeted therapies

In pleomorphic, spindle cell, and giant cell carcinoma (PSCGC) of the lung, we wondered if an integrated diagnosis including morphological and immunohistochemical features could be related to molecular status. We performed immunohistochemistry on 35 PSCGCs against TTF1, napsin A, p40, ALK, ROS1, and c-MET. Mutational status regarding EGFR, KRAS, BRAF, HER2, and PIK3CA genes was established. Of 18 PSCGCs with adenocarcinomatous or "undifferentiated" carcinoma differentiation, 8 were mutated for EGFR (n = 1), KRAS (n = 2), BRAF (n = 1), HER2 (n = 3), and PIK3CA (n = 1). No PSCGC (0/4) with only squamous cell or adenosquamous (0/2) differentiation was mutated. c-MET overexpression was only seen in PSCGC with adenocarcinomatous or undifferentiated component (n = 5) without squamous cell component. ROS1 and ALK were negative. The presence of a "targetable mutation" was correlated to the presence of morphological or immunohistochemical adenocarcinomatous differentiation (P = .0137). Integrated diagnosis of an adenocarcinomatous component in PSCGC could be associated with the presence of targetable gene mutation. Because only PSCGC with adenocarcinomatous or undifferentiated carcinoma harbors mutations, whereas PSCGC with only squamous or adenosquamous differentiation does not in our study, this might represent a prescreening for patients with PSCGC to be tested for molecular targets. Our results emphasize that careful morphological examination and the use of immunohistochemistry might be useful for the selection of PSCGC tested for a mutational target.

Abstract 5346: Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil

Abstract BACKGROUND: We have shown that Notch3 plays an important role in the regulation of esophageal squamous cell differentiation. However, it remains unclear how Notch3-mediated squamous cell differentiation influences the tumorigenicity of esophageal squamous cell carcinoma (ESCC) or its sensitivity to anti-cancer agents, including 5-Fluorouracil (5-FU). METHODS: We used 5-FU-resistant human ESCC cells, TE-11R, established by the step-wise continuous exposure of parental TE-11 cells to 5-FU. The TE-11R cells were stably transduced with intracellular domain of Notch3 (ICN3), an active form of Notch3, in a regulatable manner (Tet-On system). The anti-tumor effect of Notch3-overexpression was evaluated by implanting those cells into NOD SCID mice. 5-FU resistance was determined by calculating the 50% inhibitory concentration (IC50) of 5-FU using the WST-1 assay. Gene expression of squamous differentiation markers such as involucrin and cytokeratin 13 (CK13) as well as proliferative activity were determined. RESULTS: TE-11R cells formed bulky tumors in NOD SCID mice, and the tumor formation rate of TE-11R cells (6/6: 100%) was higher than that of TE-11 cells (1/8: 12.5%). TE-11R-derived tumors showed a less differentiated and more proliferative phenotype represented by fewer ‘keratin pearl’ formations with lower involucrin and Notch3 expressions as well as higher Ki67 expression in comparison with TE-11-derived tumors. Consistent with this, TE-11R cells showed lower gene expressions of Notch3, involucrin, and CK13, and exhibited approximately 1.4-fold higher proliferative activity than parental TE-11 cells in vitro. Notch3 overexpression resulted in the promotion of cell differentiation accompanied by increased expressions of involucrin and CK13, and suppressed cell growth in vitro as well as tumorigenicity in vivo. Finally, it clearly reduced the 5-FU resistance of TE-11R cells in vitro. IC50 values of TE-11R-ICN3 (DOX-) and TE-11R-ICN3 (DOX+) were 96.0 ± 19.7 and 21.6 ± 10.4 μM (P &amp;lt; 0.01), respectively. CONCLUSION: Our study showed that Notch3-mediated squamous cell differentiation reduced both the tumorigenicity and 5-FU resistance of ESCC cells. We suggest that a strategy to promote squamous cell differentiation may ameliorate the outcome associated with 5-FU-resistant ESCC. Citation Format: Osamu Kikuchi, Shinya Ohashi, Yukie Nakai, Yusuke Amanuma, Masahiro Yoshioka, Shin'ichi Miyamoto, Mitsuteru Natsuizaka, Hiroshi Nakagawa, Tsutomu Chiba, Manabu Muto. Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2015-5346

Expression Analysis of DGKK during External Genitalia Formation

Genetic variants in diacylglycerol kinase κ (DGKK) have been strongly associated with risk of hypospadias. We investigated the expression pattern of Dgkk during development of mouse external genitalia to better understand its function and mechanism in the etiology of hypospadias. We performed Dgkk expression analysis via indirect immunofluorescence in histological sections of CD-1 mouse embryonic and postnatal male, female and diethylsilbestrol treated external genitalia. Histological findings were supplemented with DGKK expression analysis using quantitative real-time polymerase chain reaction assays. In mouse external genitalia Dgkk was expressed in the membrane and cytoplasm of differentiating squamous epithelial cells of urethral plate/groove and epidermis but not in the undifferentiated epithelial cells of preputial lamina or basal layer of urethral groove epithelium. CD-1 gestation day 18male mouse genital tubercle treated with oil or diethylstilbestrol showed similar patterns of Dgkk expression despite many morphological differences, including formation of preputial cleft observed in diethylsilbestrol treated mice. Dgkk appears to be a marker or mediator of squamous cell differentiation during development of mouse external genitalia. However, no association exists between Dgkk expression and formation of preputial cleft in the genital tubercle of diethylsilbestrol treated mice, suggesting that these 2 events may follow independent pathways in mice. Further studies are needed to elucidate the role of DGKK in hypospadias.

Proprotein convertase 5/6A is associated with bone morphogenetic protein‐2‐induced squamous cell differentiation

Results Using a retinoic acid deficiency-induced squamous metaplasia model of HNECs, we observed a significant increase in the expression of PC5/6A, a PC member, and BMP-2, a candidate substrate for PC5/6A. Specific lentiviral shRNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Dec-RVKR-CMK, a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signalling pathways. Dec-RVKRCMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps.

Krüppel-like Factor 4 Promotes Esophageal Squamous Cell Carcinoma Differentiation by Up-regulating Keratin 13 Expression

Squamous cell differentiation requires the coordinated activation and repression of genes specific to the differentiation process; disruption of this program accompanies malignant transformation of epithelium. The exploration of genes that control epidermal proliferation and terminal differentiation is vital to better understand esophageal carcinogenesis. KLF4 is a member of the KLF family of transcription factors and is involved in both cellular proliferation and differentiation. This study using immunohistochemistry analysis of KLF4 in clinical specimens of esophageal squamous cell carcinoma (ESCC) demonstrated that decreased KLF4 was substantially associated with poor differentiation. Moreover, we determined that both KLF4 and KRT13 levels were undoubtedly augmented upon sodium butyrate-induced ESCC differentiation and G1 phase arrest. Conversely, silencing of KLF4 and KRT13 abrogated the inhibition of G1-S transition induced by sodium butyrate. Molecular investigation demonstrated that KLF4 transcriptionally regulated KRT13 and the expression of the two molecules appreciably correlated in ESCC tissues and cell lines. Collectively, these results suggest that KLF4 transcriptionally regulates KRT13 and is invovled in ESCC cell differentiation.

Genomic landscape of human papillomavirus-associated cancers.

Recent next-generation sequencing studies have generated a comprehensive overview of the genomic landscape of human papillomavirus (HPV)-associated cancers. This review summarizes these findings to provide insight into the tumor biology of these cancers and potential therapeutic opportunities for HPV-driven malignancies. In addition to the tumorigenic properties of the HPV oncoproteins, integration of HPV DNA into the host genome is suggested to be a driver of the neoplastic process. Integration may confer a growth and survival advantage via enhanced expression of viral oncoproteins, alteration of critical cellular genes, and changes in global promoter methylation and transcription. Alteration of cellular genes may lead to loss of function of tumor suppressor genes, enhanced oncogene expression, loss of function of DNA repair genes, or other vital cellular functions. Recurrent integrations in RAD51B, NR4A2, and TP63, leading to aberrant forms of these proteins, are observed in both HPV-positive head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma. Additional genomic alterations, independent of integration events, include recurrent PIK3CA mutations (and aberrations in other members of the PI3K pathway), alterations in receptor tyrosine kinases (primarily FGFR2 and FGFR3 in HPV-positive HNSCC, and ERBB2 in cervical squamous cell carcinoma), and genes in pathways related to squamous cell differentiation and immune responses. A number of the alterations identified are potentially targetable, which may lead to advances in the treatment of HPV-associated cancers.

EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition

There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.