Global gene expression profiles of human head and neck squamous carcinoma cell lines

For genomewide monitoring and identification of biomarkers of head and neck squamous cell carcinoma (HNSCC), we have conducted a systematic characterization of gene expression profiles, using human cDNA microarrays containing 9K clones, in 25 HNSCC cell lines and 1 immortalized human oral keratinocyte cell line. We used normal human oral keratinocytes (NHOKs) as a reference. Our study showed that genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in the 26 cell lines. Upregulated genes included known oncogenes, protein kinases, DNA-binding proteins and cell cycle regulators, while those commonly downregulated included differentiation markers, cell adhesion proteins, extracellular matrix proteins, structural proteins (keratins) and protease inhibitor proteins. Compared to NHOK, we observed a striking reduction in the expression of genes involved in terminal differentiation, suggesting that a loss in this process is an important signature of HNSCC. In addition, hierarchical clustering analysis as well as principal component analysis revealed 2 distinctive subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying significance analysis of microarrays, 128 genes were selected for being distinctively expressed between the 2 groups. Genes differentially expressed in the 2 subgroups include cell proliferation-related genes, IGFBP6, EGFR and VEGFC; tumor suppression and apoptosis-related genes such as Tp53, Tp63; as well as cell cycle regulators such as CCND1 and CCND2 (cyclins D1 and D2), suggesting that the 2 subgroups might have undergone different pathways of carcinogenesis.     

Low frequency of codon 61 Ha-ras mutations and lack of keratin 13 expression in 7,12-dimethylbenz[a]-anthracene—induced hamster skin tumors

Alterations in the pattern of keratin expression are a common feature of skin-tumor development. In this study, we investigated whether the loss of epidermal keratin 1 (K1) and its replacement by mucosal keratin 13 (K 13) is unique to mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), since it has been reported that human epidermal tumors do not exhibit aberrant expression of K13. With that purpose, we analyzed the keratin profiles of 16 DMBA-induced hamster skin tumors using monospecific antibodies against K1 and K13. Although all the tumors expressed K1, they also showed an overall tendency towards loss of this keratin; furthermore, none of the tumors expressed K13. Previous studies have suggested that the induction of K13 in mouse skin is related to the mutation of the Ha-ras gene by the initiating agent DMBA, a mutation consistently found in murine DMBA/TPA-induced tumors and rarely found in human skin tumors. Therefore, we also evaluated the tumors for the presence of codon-61 mutations by direct sequencing of DNA extracted from paraffin-embedded tissue sections. Only three tumors showed an A→T transversion in the second nucleotide of Ha-ras codon 61. However, presence of the mutation did not correlate with K1 staining. Although hamster skin tumors were induced by the same initiator as were mouse skin tumors, hamster skin tumors did not show the same keratin profile. Moreover, their immunohistochemical expression of K1 and K13 and their codon 61 sequences resembled that of their human counterparts. These results suggest that the aberrant expression of K13 may be unique to murine skin. Furthermore, although codon 61 Ha-ras mutation appears to be related to keratin alterations in the mouse model, this mutation is not sufficient to produce the same biochemical changes in other species.     

Functional characterization in vivo of mutant p53 molecules derived from squamous cell carcinomas of the head and neck

Loss of wild-type p53, either through deletion or mutation, has been demonstrated in most squamous cell carcinomas of the head and neck (HNSCC). Whether these mutant molecules contribute to tumor progression purely through loss of wild-type functions or by growth-promoting mechanisms, however, remains unclear. To begin to address these issues, we isolated a series of p53 cDNAs from HNSCC cell lines that contain missense or nonsense point mutations, insertions, or deletions. The ability of each of these molecules to transform NIH/3T3 cells to a malignant phenotype was assessed by stable transfection and expression under the control of a strong heterologous promoter. NIH/3T3 cells transfected with pLTR6p53, which harbors an H179L missense mutation, formed large tumors rapidly (in less than 4 wk) when transplanted to athymic mice, as did cells expressing pLTR13p53, which had undergone a V173F missense mutation and an in-frame deletion of 48 bp between codons 208 and 223. Cells transfected with pLTR17p53, predicted from the nucleotide sequence to encode a severely truncated p53 corresponding to the N-terminal 56 amino acids, also formed tumors. Cells transfected with pLTR15p53, which was predicted to encode a less severely truncated molecule, formed much smaller tumors and at lower frequencies. NIH/3T3 cells transfected with pLTR12p53 (exon 7 splice donor mutant), pLTRwtp53 (wild-type p53), or vector alone failed to form tumors for up to 2 mo after transplantation. pLTR6p53-transfected cells exhibited a highly malignant phenotype with invasion of regional lymph nodes, mediastinal and lung metastases, invasion of the abdominal wall, and dissemination throughout the peritoneal cavity. Histological asssessment of the tumors revealed intensely vascularized fibrosarcomas with numerous cellular atypia, including frequent and aberrant mitoses. Tumor explants were recultured, and northern blot analysis of cellular RNA confirmed that the expression of exogenous p53 was maintained in each case. These data indicate that different p53 mutants contribute to tumorigenesis by specific mechanisms. Furthermore, the results obtained by using the pLTR17p53 transfectants imply that some truncated molecules may overcome the effects of wild-type p53 to contribute to malignancy. Mol. Carcinog. 18:78–88, 1997. © 1977 Wiley-Liss, Inc.     

Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell–cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.     

连环蛋白ｐ１２０在胰腺癌细胞株中的表达及其意义

目的：了解连环蛋白ｐ１２０（ｐ１２０ｃｔｎ）及ＶＥＧＦ在胰腺癌细胞株中的表达情况,探讨ｐ１２０ｃｔｎ对胰腺癌细胞生物学行为的影响。方法：采用ＲＴ-ＰＣＲ、Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ方法检测５株胰腺癌细胞及胰腺星状细胞中ｐ１２０ｃｔｎ及ＶＥＧＦ的表达情况;选取表达差异明显的ｐａｎｃ １及Ｐａｔｕ８９８８细胞株,利用细胞侵袭迁移试验观察其生物学行为。结果：与胰腺星状细胞相比,胰腺癌细胞株中ｐ１２０ｃｔｎ的表达均上调,但在不同胰腺癌细胞株中表达存在差异;在同一细胞株中,ｐ１２０ｃｔｎ与ＶＥＧＦ的表达量呈正相关;高表达ｐ１２０ｃｔｎ的胰腺癌细胞,其侵袭迁移能力要明显低于低表达的细胞。结论：胰腺癌细胞株中ｐ１２０ｃｔｎ的表达明显上调,并与ＶＥＧＦ的表达存在相关性,胰腺癌细胞ｐ１２０ｃｔｎ高表达其侵袭转移能力可能下降。     

酪氨酸蛋白激酶受体EphA2及其配体EFNA1在23种肿瘤细胞系中的表达及其意义

背景与目的：酪氯酸蛋白激酶受体EphA2及其配体EFNA1可能在肿瘤的发生发展过程中起着重要的作用。本研究探讨EphA2／EFNA1在不同肿瘤细胞中的表达及其意义。方法：用RT-PCR方法，检测了23种人类肿瘤细胞系中EphA2／EFNA1在mRNA水平上的表达，、并用Western blot方法检测了EphA2蛋白在不同肿瘤细胞系中的表达水平。结果：RT—PCR结果显示．除白血病细胞K562和HL60，以及视网膜母细胞瘤RB几乎检测不到EphA2／EFNA1外．EphA2／EFNA1高表达于肝癌、肺癌、卵巢癌、官颈癌、前列腺癌、胃腺癌、脑胶质瘤、膀胱癌、成骨肉瘤及恶性黑色素瘤等20种细胞系中。Western blot结果显示，除K562外，EphA2蛋白在不同肿瘤细胞系中均有表达。结论：EphA2／EFNA1高表达于人类多种肿瘤细胞中，并且EFNA1的表达水平相对低于EphA2的表达、EphA2．／EFNA1可作为一个较为广潜的肿瘤标记，并有可能成为肿瘤治疗的靶点。     

In vitro chemotherapy sensitivity patterns of human colon carcinoma continuous cell lines

The cells obtained from four established human colon carcinoma cell lines were cultured in soft agar in the presence of eight chemotherapeutic agents that have been used clinically in patients with metastatic colorectal carcinoma. 5-Fluorouracil, mitomycin C, adriamycin, and cis-platinum were highly cytotoxic in vitro at the concentrations employed. These findings were in marked contrast to in vitro chemotherapy sensitivity patterns previously reported by us for a large series of primary human colorectal carcinomas cultured in soft agar. The possible reasons behind these differences are discussed.     

Involvement of VILIP-1 (visinin-like protein) and opposite roles of cyclic AMP and GMP signaling in in vitro cell migration of murine skin squamous cell carcinoma

VILIP-1 (visinin-like protein 1) is downregulated in various human squamous cell carcinoma (SCC). In a mouse skin SCC model VILIP-1 expression is reduced in aggressive tumor cells, accompanied by reduced cAMP levels. Overexpression of VILIP-1 in aggressive SCC cells led to enhanced cAMP production, in turn causing a reduction in invasive properties. Moreover, in primary neurons and neuronal tumor lines VILIP-1 enhanced cGMP signaling. Here, we set out to determine whether and how cAMP and cGMP signaling contribute to the VILIP-1 effect on enhanced SCC model cell migration, and thus most likely invasiveness in vivo. We found stronger increase in cGMP levels in aggressive, VILIP-1-negative SCC cells following stimulation of guanylyl cyclases NPR-A and -B with the natriuretic peptides ANP and CNP, respectively. Incubation with ANP or 8Br-cGMP to increase cGMP levels further enhanced the migration capacity of aggressive cells, whereas cell adhesion was unaffected. Increased cGMP was caused by elevated expression levels of NPR-A and -B. However, the expression level of VILIP-1 did not affect cGMP signaling and guanylyl cyclase expression in SCC. In contrast, VILIP-1 led to reduced migration of aggressive SCC cells depending on cAMP levels as shown by use of adenylyl cyclase (AC) inhibitor 2',3'-dideoxyadenosine. Involvement of cAMP-effectors PKA and EPAC play a role downstream of AC activation. VILIP-1-positive and -negative cells did not differ in mRNA expression of ACs, but an effect on enhanced protein expression and membrane localization of ACs was shown to underlie enhancement of cAMP production and, thus, reduction in cell migration by VILIP-1.     

Oral and Oropharyngeal squamous cell carcinoma: prognostic and predictive parameters in the etiopathogenetic route

Introduction: Oral and oropharyngeal squamous cell carcinoma (OSCC and OPSCC) represents an increasing problem in the global public health. Indeed, squamous cell carcinoma is the most frequent malignancy in oral cavity and 1 of the 10 most common cancers worldwide. According to the most recent GLOBOCAN estimate in Europe between 2012 and 2015, there was an overall increasing incidence and mortality for oral cancer, mostly HPV-related in the oropharyngeal region with evidence of significant differences from the prognostic and therapeutic point of view.

Areas covered: Until now, the management of the patients is based on classical histologic parameters such as TNM and tumor grading, but new molecular and cell markers have been investigated to improve patients’ treatment and survival. Therefore, there is a need for new biomarkers characterizing the cancer diversity, with the consequent possibility of patient stratification for specific treatment.

Expert commentary: This review aims to discuss some of the most relevant and novel genetic, epigenetic, and histological prognostic biomarkers in oral cancer, highlighting the main differences between HPV-unrelated oral squamous cell carcinoma (OSCC) and HPV-related oropharyngeal squamous cell carcinoma (OPSCC) that may aid in stratifying prognostic subgroups and rationalizing treatment decisions.     

Differential sensitivity of paclitaxel-induced apoptosis in human esophageal squamous cell carcinoma cell lines

Purpose: Paclitaxel is a highly effective chemotherapy agent against adenocarcinomas and squamous cell carcinomas of the esophagus. However, its precise effects in human esophageal cancer cells are not well understood. This study was designed to examine the relationship between cell-cycle phases of paclitaxel-activated checkpoints and to elucidate the molecular pathway of the effect of paclitaxel in human esophageal squamous cell carcinoma (ESCC) cell lines. Methods: The three human ESCC cell lines—TE-2, TE-13 and TE-14—were examined for their response to paclitaxel. ESCC cells were treated with various concentrations of paclitaxel for 1–3 days using MTT assay. The cell-cycle progression and apoptosis were examined by flow cytometry. DNA fragmentation assay was carried out to confirm the fragmented cells as hallmark for apoptotic cells. In additional, the expression of apoptosis-related proteins in ESCC-treated cells was then examined by Western blot analysis. Results: TE-14 cells demonstrated the highest sensitivity among all cells. G2/M cell-cycle arrest occurs prior to paclitaxel-induced apoptosis in ESCC cells. The fragmentation of chromatin was observed in drug treated TE-13 and TE-14 cells by flow cytometry and DNA ladder formation. In contrast, the measurement for TE-2 cells was more suggestive of phenotype a resistant in response to paclitaxel treatment. Western blot analysis results showed that the mitochondrial pathway might be involved in paclitaxel-induced apoptosis in ESCC cell lines. Conclusion: Differential sensitivity was observed in human ESCC cell lines in response to paclitaxel treatment. G2/M arrest occurs with a prior to paclitaxel-induced apoptosis and might be mediated by the mitochondrial (intrinsic) apoptosis pathway in human ESCC cells. A. Faried and L.S. Faried contributed equally to this project and are considered co-first authors.     

Antitumor activity of suberoylanilide hydroxamic acid against human oral squamous cell carcinoma cell lines in vitro and in vivo

It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.