Ming-Sound Tsao

ras mutation and expression of the ras-regulated genes osteopontin and cathepsin L in human esophageal cancer

As part of our ongoing studies to characterize molecular alterations in a well‐defined series of surgically resected esophageal cancers, we examined the expression of 2 ras‐regulated genes, whose products (osteopontin and cathepsin L) previously were shown to be associated with tumor invasion and metastasis. RNA was extracted from primary esophageal tumors (adenocarcinomas, 19; squamous‐cell carcinomas, 6) and matched histologically normal esophageal mucosa from the distant resection margin. Northern analysis was used to quantitate RNA, relative to an 18S rRNA control, and immunohistochemistry to assess the tissue distribution of osteopontin. In addition, H‐, K‐ and N‐ras mutations were studied in the same tissues using PCR and hybridization with allele (mutant)‐specific oligonucleotide probes. We demonstrated a K‐ras mutation (codon 12, GTT) in one esophageal adenocarcinoma. The ras‐regulated gene osteopontin was over‐expressed in 100% of squamous‐cell carcinomas and in 58% of adenocarcinomas relative to matched normal esophageal mucosa. Patterns of immunoreactivity for osteopontin protein also varied between squamous‐cell carcinomas (tumor cell staining) and adenocarcinomas (predominantly tumor‐infiltrating macrophages). Expression of cathepsin L also varied with esophageal tumor histology, with over‐expression in 58% of primary esophageal adenocarcinomas and 33% of squamous‐cell cancers. Int. J. Cancer 72:739–745, 1997.

Tight junction protein zo‐2 is differentially expressed in normal pancreatic ducts compared to human pancreatic adenocarcinoma

Differential display of hamster mRNA identified a fragment present in normal pancreatic duct cells that is not expressed in pancreatic duct carcinoma cells. Sequence analysis showed an 88% and 82% identity, respectively, to the cDNA of the canine and human tight junction zo‐2 gene. Semi‐quantitative RT‐PCR analysis of human ZO‐2 revealed a striking difference in the expression of various regions of the ZO‐2 transcript in normal and neoplastic cells and the presence of an abnormality at the 5′‐end of mRNA. RACE analysis identified 2 human ZO‐2 mRNAs that encode proteins of different lengths, designated as ZO‐2A and ZO‐2C. The difference between the 2 forms of ZO‐2 is the absence of 23 amino acid residues at the N terminus of ZO‐2C compared with ZO‐2A. Although ZO‐2C was expressed in normal pancreatic cells and a majority of neoplastic tissues analyzed, ZO‐2A was undetectable except in one case in all of the pancreatic adenocarcinomas analyzed. This suggests the presence of a yet to be identified motif important for cell‐growth regulation within the 23–amino acid residue N‐terminal peptide of ZO‐2A, MPVRGDRGFPPRRELSGWLRAPG. Int. J. Cancer 82:137–144, 1999.

Targeting NAD(P)H:Quinone oxidoreductase (NQO1) in pancreatic cancer

NAD(P)H:Quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species. The aims of our study were to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated inceased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage‐independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months.

A sequence-based approach to identify reference genes for gene expression analysis

BackgroundAn important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate.MethodsSerial analysis of gene expression (SAGE) profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR), and their impact on differential expression analysis of microarray data was evaluated.ResultsWe show that (i) conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii) reference genes identified for lung cancer do not perform well for other cancer types (breast and brain), (iii) reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv) normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung cancer exhibit higher statistical significance using a dataset normalized with our reference genes relative to normalization without using our reference genes.ConclusionsOur analyses found NDUFA1, RPL19, RAB5C, and RPS18 to occupy the top ranking positions among 15 suitable reference genes optimal for normalization of lung tissue expression data. Significantly, the approach used in this study can be applied to data generated using new generation sequencing platforms for the identification of reference genes optimal within diverse contexts.

zo-2 gene alternative promoters in normal and neoplastic human pancreatic duct cells

We have observed that 2 forms of zonula occludens 2 (ZO‐2) protein, ZO‐2A and ZO‐2C, are expressed in normal human pancreatic duct cells, but only ZO‐2C in pancreatic duct adenocarcinoma. We report here partial organization of the zo‐2 gene. Transcription of 2 forms of ZO‐2 mRNA is driven by alternative promoters PA and PC. Lack of expression of ZO‐2A in neoplastic cells is caused by inactivation of the downstream promoter PA. Analysis of the promoter PA sequence and function in normal and neoplastic cells demonstrated that neither structural changes (mutations) nor a change in the pool of transcription factors is responsible for its inactivation. Although hypermethylation was found in a large number of cancer clones, treatment with 5‐aza‐2′‐deoxycytidine did not fully cause the promoter function to recover. We conclude that the initial down‐regulation of zo‐2 promoter PA activity in pancreatic duct carcinomas is due to the structural or functional alteration(s) in the regulatory elements, localized outside the analyzed promoter region. Methylation of PA is responsible for the inactivation of the suppressed promoter at the late stages of tumor development. Int. J. Cancer 83:349–358, 1999.

Role of Rac1-dependent NADPH oxidase in the growth of pancreatic cancer

K-ras mutations occur in as high as 95% of patients with pancreatic cancer. K-ras activates Rac1-dependent NADPH oxidase, a key source of superoxide. Superoxide has an important function in pancreatic cancer cell proliferation, and scavenging or decreasing the levels of superoxide inhibits pancreatic cancer cell growth both in vitro and in vivo. DNA microarray analysis and RT–PCR has demonstrated that Rac1 is also upregulated in pancreatic cancer. The aim of this study was to determine whether inhibiting Rac1 would alter pancreatic tumor cell behavior. Human pancreatic cancer cells with mutant K-ras (MIA PaCa-2), wild-type K-ras (BxPC-3) and the immortal H6c7 cell line (pancreatic ductal epithelium) expressing K-ras oncogene (H6c7eR-KrasT) that is tumorigenic, were infected with a dominant/negative Rac1 construct (AdN17Rac1). In cells with mutant K-ras, AdN17Rac1 decreased rac activity, decreased superoxide levels and inhibited in vitro growth. However, in the BxPC-3 cell line, AdN17Rac1 did not change rac activity, superoxide levels or in vitro cell growth. Additionally, AdN17Rac1 decreased superoxide levels and inhibited in vitro growth in the KrasT tumorigenic cell line, but had no effect in the immortalized H6c7 cell line. In human pancreatic tumor xenografts, intratumoral injections of AdN17Rac1 inhibited tumor growth. These results suggest that activation of Rac1-dependent superoxide generation leads to pancreatic cancer cell proliferation. In pancreatic cancer, inhibition of Rac1 may be a potential therapeutic target.

Induction chemoradiotherapy facilitates radical resection of T4 non-small cell lung cancer invading the spine.

OBJECTIVE We evaluated the outcome, long-term results, and factors affecting outcome of induction chemoradiotherapy followed by surgical resection for T4 non-small cell lung cancer invading the spine. METHODS Retrospective analysis of 23 consecutive patients undergoing radical vertebral resection for non-small cell lung cancer invading the spine between 1996 and 2007 was performed. In most cases, induction chemoradiotherapy consisted of cisplatin and etoposide followed by 45 Gy of radiation. Surgical resection with vertebrectomy was performed en bloc in either a 1-stage or 2-stage operation. Survival was estimated by Kaplan-Meier techniques. The log-rank comparison was used to compare groups. RESULTS There were 13 men and 10 women with a median age of 61 years (range 32-75). Twenty-two patients had induction chemoradiotherapy and 1 had induction chemotherapy alone. Vertebral resections included 6 total vertebrectomies, 15 hemivertebrectomies, and 2 partial vertebrectomies. Complete resection was achieved in 19 (83%) patients. Two (8.7%) patients died postoperatively. Pathologic complete response was observed in 10 (43%) patients. The 3-year survival was 58% (median follow-up, 34 months). Patients who achieved pathologic complete response or near complete response (viable tumor cells < 1%) demonstrated significantly better survival than those who did not (3-year survival, 92% vs 20%; P = .006). CONCLUSION Highly selected patients with lung cancer invading the spine can potentially be cured with induction chemoradiation therapy followed by radical en bloc resection of the tumor. A multidisciplinary operative strategy allows a significant chance of achieving complete resection in patients requiring multilevel hemivertebrectomy or total vertebrectomy and an appreciable cure rate.

The Role of Cancer-Testis Antigens as Predictive and Prognostic Markers in Non-Small Cell Lung Cancer

Background Cancer-Testis Antigens (CTAs) are immunogenic proteins that are poor prognostic markers in non-small cell lung cancer (NSCLC). We investigated expression of CTAs in NSCLC and their association with response to chemotherapy, genetic mutations and survival. Methods We studied 199 patients with pathological N2 NSCLC treated with neoadjuvant chemotherapy (NAC; n = 94), post-operative observation (n = 49), adjuvant chemotherapy (n = 47) or unknown (n = 9). Immunohistochemistry for NY-ESO-1, MAGE-A and MAGE-C1 was performed. Clinicopathological features, response to neoadjuvant treatment and overall survival were correlated. DNA mutations were characterized using the Sequenom Oncocarta panel v1.0. Affymetrix data from the JBR.10 adjuvant chemotherapy study were obtained from a public repository, normalised and mapped for CTAs. Results NY-ESO-1 was expressed in 50/199 (25%) samples. Expression of NY-ESO-1 in the NAC cohort was associated with significantly increased response rates (P = 0.03), but not overall survival. In the post-operative cohort, multivariate analyses identified NY-ESO-1 as an independent poor prognostic marker for those not treated with chemotherapy (HR 2.61, 95% CI 1.28–5.33; P = 0.008), whereas treatment with chemotherapy and expression of NY-ESO-1 was an independent predictor of improved survival (HR 0.267, 95% CI 0.07–0.980; P = 0.046). Similar findings for MAGE-A were seen, but did not meet statistical significance. Independent gene expression data from the JBR.10 dataset support these findings but were underpowered to demonstrate significant differences. There was no association between oncogenic mutations and CTA expression. Conclusions NY-ESO-1 was predictive of increased response to neoadjuvant chemotherapy and benefit from adjuvant chemotherapy. Further studies investigating the relationship between these findings and immune mechanisms are warranted.

REGULATION OF PANCREATIC CANCER GROWTH BY SUPEROXIDE

K‐ras mutations have been identified in up to 95% of pancreatic cancers, implying their critical role in the molecular pathogenesis. Expression of K‐ras oncogene in an immortalized human pancreatic ductal epithelial cell line, originally derived from normal pancreas (H6c7), induced the formation of carcinoma in mice. We hypothesized that K‐ras oncogene correlates with increased non‐mitochondrial‐generated superoxide (O  2.− ), which could be involved in regulating cell growth contributing to tumor progression. In the H6c7 cell line and its derivatives, H6c7er‐Kras+ (H6c7 cells expressing K‐ras oncogene), and H6c7eR‐KrasT (tumorigenic H6c7 cells expressing K‐ras oncogene), there was an increase in hydroethidine fluorescence in cell lines that express K‐ras. Western blots and activity assays for the antioxidant enzymes that detoxify O  2.− were similar in these cell lines suggesting that the increase in hydroethidine fluorescence was not due to decreased antioxidant capacity. To determine a possible non‐mitochondrial source of the increased levels of O  2.− , Western analysis demonstrated the absence of NADPH oxidase‐2 (NOX2) in H6c7 cells but present in the H6c7 cell lines expressing K‐ras and other pancreatic cancer cell lines. Inhibition of NOX2 decreased hydroethidine fluorescence and clonogenic survival. Furthermore, in the cell lines with the K‐ras oncogene, overexpression of superoxide dismutases that detoxify non‐mitochondrial sources of O  2.− , and treatment with the small molecule O  2.− scavenger Tempol, also decreased hydroethidine fluorescence, inhibited clonogenic survival and inhibited growth of tumor xenografts. Thus, O  2.− produced by NOX2 in pancreatic cancer cells with K‐ras, may regulate pancreatic cancer cell growth.

A Study of Smoking, p53 Tumor Suppressor Gene Alterations and Non-Small Cell Lung Cancer

PURPOSE The purpose of this study was to investigate the relationship between smoking and p53 tumor suppressor gene alterations, and their association with clinicopathologic features and prognosis in non-small cell lung cancer (NSCLC). METHODS For 111 of 119 stage I-III NSCLC patients that had been followed prospectively, tumor p53 protein accumulation was measured immunohistochemically (IHC). Staining was evaluated as a score (p53IHCS) combining intensity and percent distribution. RESULTS Forty-eight of 111 (43%) tumors had p53IHCS > 1. p53 IHC was associated with increasing tumor size (T) (p = 0.035), nodal status (N) (p = 0.091), stage (p = 0.054), and histology: squamous cell carcinoma (70%) and adenocarcinoma (27%) (p = 0.0002). In logistic regression analysis, p53 IHC was associated with squamous cell histology versus other histotypes [adjusted odds ratio (OR)5.90, 95% confidence interval (CI) 2.34-14.90]. p53 IHC was not associated with smoking variables. In multivariate proportional hazards analysis, p53IHCS and pack-years smoked (PY), both as continuous variables, were negative prognostic factors. The adjusted hazard ratios (HR) for the survival outcome recurrence for p53IHCS and PY were 1.20 (95% CI 1.02-1.40) and 1.03 (95% CI 1.01-1.04), and for death due to recurrent disease (DRD) were 1.35 (95% CI 1.11-1.64) and 1.03 (95% CI 1.01-1.04), respectively. Comparing the 75th percentile to the baseline 0, the adjusted HR for p53IHCS (5 vs. 0) was 4.5 and for PY (55 vs. 0) was 5.1 for the outcome DRD. Both variables demonstrated a dose-response relationship with survival. CONCLUSIONS PY and p53IHCS are significant, independent and important predictors of recurrence and DRD in stage I-III NSCLC.