Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology. / Dissertation

The esophageal squamous cell carcinoma (ESCC) is known to be one of the most difficult malignancies to treat among digestive carcinomas. The esophageal squamous cell carcinoma and adenocarcinoma are the two most common cell types of esophageal cancer in the world. Even though the incidence of esophageal adenocarcinoma has been dramatically increasing in Western populations during the past two decades, esophageal squamous cell carcinoma remains the predominant type of esophageal malignancy in the remainder of the world. In Taiwan, ESCC is the ninth leading cause of cancer deaths, the 6th among male. In spite of advances in surgical techniques and perioperative management in recent decades, current modalities of therapy for this disease still offer poor survival and cure rates. Even in resectable diseases, the 5-year survival rates were only less than 20%. Recently, esophageal squamous cell carcinoma and adenocarcinoma were increasingly recognized as two entities with different biologic behavior and outcome. Consequently, the surgical risks and oncologic benefits of esophagectomies for esophageal squamous cell carcinoma patients are controversial and not confessed. From 1991 to 2003, 216 esophageal squamous cell carcinoma patients underwent esophagectomy were enrolled and analyzed retrospectively. Among these patients, 166 patients underwent transthoracic esophagectomy and 50 patients underwent transhiatal esophagectomy. The overall hospital mortality and postoperative complication rates were 9.7% and 49%, respectively. The overall 5-year survival rate was 16.8%. The hospital mortality rate, postoperative complication rate, length of hospital stay and amount of intra-operative blood loss or transfusion were not significantly different between both groups. But, shorter operative time was noticed in transhiatal group (p<0.001). Patients underwent either transthoracic or transhiatal esophagectomy had comparable long-term survival. The pTNM stage was independent prognostic factors for patients underwent transthoracic esophagectomy. However, location of tumors (p=0.0085) and pathologic tumor length (p=0.0118) were significant predictors for patients underwent transhiatal esophagectomy. In this part of study, we found that both transthoracic and transhiatal esophagectomies could provide comparable survival benefits for esophageal squamous cell carcinoma patients. However, the traditional pTNM staging system might underestimate the severities of ESCC patients who underwent transhiatal esophagectomy. Due to the dismal results of ESCC patients after surgery and the findings of multi-environmental and/or genetic factors involved in the carcriogenesis of ESCC, further realizing the molecular mechanisms of carcinogens in the development of esophageal squamous cell carcinoma is crucial for prevention and treatment for this disease. Epidemiological analysis showed that the prevalence of esophageal squamous cell carcinoma varied in different geographic areas. The various prevalence of disease in different parts of the world reflects different forms and extents of exposure to these etiological agents involving the development of this disease. In stead of tobacco and alcohol, recent reports indicated that betel quid (BQ) chewing also significantly correlated with the occurrence of esophageal squamous cell carcinoma in Taiwanese. The mechanisms behind the BQ-related esophageal squamous cell carcinoma in Taiwan are worthy for further investigation. Previous studies have shown that certain carcinogens may induce a ¡§fingerprint-like¡¨ ¡V like pattern of mutations at the p53 gene, both in terms of mutation type and codon specificity. However, the role of p53 mutation in the etiology of esophageal squamous cell carcinoma has not been rigorously studied in Taiwan. The incidence of p53 mutations in ESCC associated with risk factors has not been explored in Taiwanese. Accordingly, 75 primary esophageal squamous cell carcinoma specimens were collected for examining the incidence of mutations in the conserved regions of p53 gene by using polymerase chain amplification and direct sequencing of amplified products. There were 37 mutations of p53 gene detected in 45.5% (34/75) of tumor specimens. These mutations significantly clustered in exon 5 (21/37) of p53 gene. The incidence of p53 mutations didn¡¦t associate with clinicopathological characteristics and habits of cigarette smoking or alcohol drinking. However, BQ chewer exhibited significantly higher incidence of p53 gene mutations than non-chewer (67.6% vs. 32.4%, p=0.007). After controlling confounding factors of cigarette smoking and alcohol drinking, BQ chewing still showed significant impact on the incidence of p53 mutation in esophageal squamous cell carcinomas (RR=4.233; 95% CI, 1.317-13.603). The A:T to G:C transition (8/37, 21.6%) and G:C to T:A transversion (5/23, 13.5%) were the prevalent spectrum of p53 gene mutations. All A:T to G:C transitional mutations occurred in patients with habits of betel quid chewing and cigarette smoking. Noticeably, alcohol drinking could enhance this peculiar spectrum of p53 mutation in esophageal squamous cell carcinoma. Therefore, p53 gene might be one of the molecular targets of betel quid carcinogens in the development of esophageal squamous cell carcinoma in Taiwanese. Determination of the importance of p53 gene mutation in ESCC requires further study. To elucidate the role of cell cycle associated genes in ESCC, 40 primary esophageal squamous cell carcinoma patients were included in this part of study. Tissue samples were analyzed for cell proliferation, DNA content, mutation of p53 gene, and expression of p16, p21waf1/cip1, pRb and p53 proteins. In this part of study, 75% of tumors exhibited aneuploid DNA content. Significantly higher S-phase fractions were detected in tumor samples (p<0.001). The p53 immunostaining was detected in 62.5% (25/40) of tumor tissues and 50% of tumors were p21waf1/cip1 overexpression. The p16 protein was only detected only in 8 of the 40 ESCC (20.0%) tissue samples by immunohistochemistry. The nucleus stained Rb protein was detected in 38 ESCC tissue samples and all of them were phosporylated status. The phosphorylation of pRb at Ser-795, Ser-789 and Ser-807/811 was detected in 87.5% (35/40), 72.5 (29/40) and 42.5% (17/40) of ESCC tissue samples, respectively. Expression of p16 protein, total pRb or phospho-Rb expression status did not correlate with the clinicopathological parameters of patients. The overexpression of p21waf1/cip1 protein didn¡¦t correlate with p53 gene status, but significantly correlated with the existence of abnormal DNA content (P=0.028). Advanced pTNM stage, lymph node metastasis and p21waf1/cip1 overexpression conferred survival disadvantages in univariate analysis (P=0.013, 0.045 and 0.017, respectively). A Cox multivariable analysis revealed pTNM stage (IIB/III/IV vs. I/IIA; p=0.024) associated with p21waf1/cip1 overexpression (positive vs. negative; p=0.035) as independent prognostic factors in esophageal squamous cell carcinomas. Surprisingly, p21waf1/cip1 overexpression significantly compromised the survival of patients with mutated p53 gene (p=0.035). However, no significant dismal effect of p21waf1/cip1 overexpression can be seen in patients with wild-type p53 gene (P=0.175). Consequently, overexpression of p21waf1/cip1 is correlated with chromosomal instability and serves as an adverse prognostic predictor for esophageal squamous cell carcinoma patient. Its dismal effect is more prominent when p53 gene is mutated. The ribonucleotide reductase (RNR) is an S phase-specific dimeric enzyme and is the rate-limiting enzyme of DNA synthesis pathway responsible for the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides (dNTPs), which are the building blocks for DNA replication and repair in all living cells. The RNR enzyme was formed by the association of RRM1 and RRM2 subunits. Normally, the levels of RRM2 expression modulate the RNR enzymatic activity. However, RRM2 also plays an important role in other aspects of the malignant phenotype such as tumor development and drug resistance. Recently, the p53-inducible ribonucleotide reductase small subunit homologue, p53R2, has been isolated and shown to play a crucial role in DNA repair after DNA damage. However, the function of p53R2 is still unclear especially in tumor cells. By immunohistochemistry, the expression of RRM2 and p53R2 proteins were detected in 94.1% (80/85) and 55.3% (47/85) of ESCCs tissue samples, respectively. No significant correlation could be found between RRM2 protein expression and gender, depth of tumor invasion, lymph-node involvement and pTNM stage. The p53R2 expression status also did not correlate with the gender of patients and the depth of tumor invasion. However, the presence of p53R2 protein expression significantly correlated with pTNM stages of tumors (p=0.027) and lymph node metastasis (p=0.009). In Cox multivariable regression analysis, p53R2 expression (positive vs. negative; p=0.011, HR: 3.096, 95% CI: 1.294-7.407) together with pTNM stage (IIB/III/IV vs. I/IIA; p=0.005, HR: 2.496, 95% CI: 1.320-4.719) was shown to have independent prognostic impact on survival of ESCC patients. Accordingly, in the analysis of cell cycle associate genes, the p53 mutations associated with loss of Rb pathway function would be the critical event in the development of human esophageal squamous cell carcinoma. In ESCC, loss of p53 pathway function is attributable to p53 mutations. However, the Rb pathway might be perturbed by inactivation of p16 and over-expressed p21waf1/cip1 protein in ESCC tissues. Consequently, the perturbed Rb function results in up-regulation ribonucleotide reductase activity, causing DNA precursors overproduction and cell proliferation. Using immunohistochemistry, our findings provide the first evidence of RRM2 and p53R2 expression in human ESCCs. We identified the different prognostic effects of RRM2 and p53R2 expression in human ESCCs. The identification of different roles of p53R2 and RRM2 involved in the carcinogenesis of esophageal squamous cell carcinomas might be useful for designing more effective RRM2 or p53R2 specific target therapy for esophageal squamous cell carcinoma to improve the clinical outcome of patients with esophageal carcinoma.

DNA repair mechanisms counteract the formation of deleterious DNA lesions and maintain genomic integrity. Nucleotide excision repair (NER) is an important DNA repair pathway because of its extraordinarily large substrate specificity. P53 protein regulates NER pathway in a transcription-dependent or transcription-independent manner. Inherited polymorphisms of NER pathway genes (XPC, HR23B, XPA, DDB2, XPB, XPD, ERCC1, XPF, and XPG) and TP53 gene may contribute to individual variations in genetic susceptibility to OSCC and correlate with the prognosis of 204 OSCC patients. We carried out a hospital-based case-control study to investigate the association of 25 various polymorphisms of nine NER pathway genes and TP53 gene with the risk for OSCC. There were 34 newly diagnosed OSCC patients and 135 frequency-matched controls without BQ chewing and smoking habit as well as 313 newly diagnosed OSCC patients with BQ chewing or smoking habit and 312 frequency-matched controls being recruited between November 2003 and July 2007 at Kaoshiung Veterans General Hospital. Genotyping was performed using the PCR-RFLP techniques or TaqMan real-time PCR method. The significant association between polymorphisms of NER pathway genes and OSCC risk was mainly found among subjects with BQ chewing or smoking habit. In the single locus analysis, GA and AA genotypes of ERCC1 G-641A (AOR, 0.64; 95% CI, 0.45-0.93 and AOR, 0.48; 95% CI, 0.29-0.79, respectively; p for trend, 0.002), CT genotype of XPF C-850T (AOR, 1.53; 95% CI, 1.08-2.18; p for trend, 0.014), as well as GG genotype of XPB A-1039G (AOR, 0.51; 95% CI, 0.26-0.98; p for trend 0.034) were significantly associated with the risk of OSCC. Furthermore, -641G/ -425T or -641G/ -425C haplotype of ERCC1 (AORs, 1.34; 95% CI, 1.02-1.77 and AOR, 1.56; 95% CI, 1.18-2.07, respectively; p for trend 0.002) as well as -850T/ -247T and -850T/ -247C haplotype of XPF (AOR, 1.45; 95% CI, 1.09-1.94 for; p for trend 0.016) were strongly associated with the risk of OSCC. A trend toward increased risk of OSCC was observed when people with the increasing number of at risk genotypes in the combined analyses of nine NER pathway genes with (p for trend, <0.001) or without (p for trend 0.001) TP53 gene. Finally, in the stratification analysis, the combined effects of nine NER pathway genes had a significantly increased risk of OSCC among younger group (¡Ø50 years old), Fukienece population, BQ chewers, light smokers, or light drinkers. Besides, in the prognosis analysis of 204 OSCC patients, HR23B A-823C, polymorphisms of XPA gene, XPD C-643G, XPG C787G, and the number of at risk genotypes of NER pathway genes were associated with pathologic stage, T classification, or N classification. The association between NER genetic polymorphisms and survival of patients was only found in XPA C-1778T polymorphism. These results suggested that the single polymorphism of XPB A-1039G, ERCC1 G-641A and XPF C-850T, the joint effect of genetic polymorphisms of NER pathway genes, and gene-environment combined effect were associated with the risk of OSCC. Furthermore, in the analysis of NER genetic polymorphisms and prognosis of OSCC, we found polymorphisms of XPA gene might be a prognostic factor for OSCC.

Diss. (sammanfattning)--Stockholm : Karolinska institutet, 2009.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Surgery / Master / Master of Research in Medicine

published_or_final_version / Medicine / Master / Doctor of Medicine

No description available.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.