Alterations in p53 and pRb pathways and their prognostic significance in oesophageal cancer

doi:10.1016/S0959-8049(02)00007-2

European Journal of Cancer

Volume 38, Issue 6, 2002, Pages 832-841

https://doi.org/10.1016/S0959-8049(02)00007-2 Get rights and content

Abstract

The pRb (p16-pRb-cyclin D1) and p53 (p53-MDM2-p21) pathways play a critical role in tumorigenesis. To evaluate which of these cell cycle regulatory proteins are related to patients' prognosis, a comprehensive analysis of alterations in these components was carried out in 100 ESCCs (oesophageal squamous cell carcinoma) using immunohistochemistry and correlated with clinicopathological parameters by univariate analysis. Overexpression of p53, MDM2 and cyclin D1 proteins was observed in 73, 42 and 67% of the cases, respectively, while loss of expression of p21, p16 and pRb was observed in 36, 45 and 75% of the cases, respectively. Multiple logistic regression analysis revealed that loss of p16 immunoreactivity was a significant risk factor for tumour stage (pT) (Odds Ratio (OR)=3.3), whereas the loss of pRb was a significant risk factor for nodal metastasis (pN) (OR=8.8). MDM2 overexpression emerged as the most significant risk factor for distant organ metastasis (pM) (OR=4.6). Of the ESCC patients who underwent oesophagectomy, 50 cases were followed-up for a maximum period of 44 months and median of 16 months. Survival analysis revealed that Cyclin D1 overexpression is an adverse prognosticator for disease-free survival, as well as overall survival, and tumour stage (pT) is an adverse prognosticator for disease-free survival. In conclusion, these data support a model of oesophageal cancer pathogenesis in which both the pRb and p53 pathways are inactivated and suggests an in-depth evaluation of the clinical utility of these putative markers is warranted.

Introduction

Despite advances in therapeutic strategies and postoperative management, prognosis for oesophageal cancer patients remains poor. Improvements in the prognosis and surveillance await a more comprehensive understanding of the molecular alterations and clinicopathological characteristics as predictors of a parsimonious model in tumour control. The clinical characterisation of oesophageal carcinoma remains inadequate using the conventional histological staging system. The TNM staging for oesophageal cancer [1] considered as the most important prognostic indicator does not differentiate between tumours confined to the mucosa and those involving the submucosa, a distinction that has been shown to be of considerable prognostic significance [2]. Several studies have demonstrated considerable heterogeneity in prognosis among oesophageal squamous cell carcinomas (ESCC) of various histological types. Submucosal oesophageal cancer has been shown to be associated with a significant risk of vascular invasion and lymph node metastasis [3]. Current inadequacy of clinicopathological parameters as prognosticators warrants a clearer understanding of the molecular alterations in predicting prognosis of the disease. Various molecular biological factors have been proposed as prognostic indicators of oesophageal cancer [4]. Cell cycle regulators of prognostic relevance in oesophageal cancer include, mutations in the tumour suppressor gene TP53, allelic loss at chromosomal loci encoding p16 and pRb and altered expression of p21, cyclin D1 and MDM2 5, 6, 7. Nevertheless, nearly all of these studies have examined these markers in isolation or in dual combinations and therefore the overall conclusions from these studies are discordant and a composite picture of the interactions between p53-MDM2-p21 and p16-pRb-cyclin D1 pathways is currently lacking.

The aetiological factors implicated in the pathogenesis of ESCCs are markedly different in the Western population compared with that of developing countries such as India. In Western countries, alcohol consumption and tobacco smoking are the major predisposing factors for ESCCs while, in the developing countries such as India and China, tobacco chewing and a variety of dietary factors confounded by nutritional deficiency are causally associated with the disease 8, 9, 10. The exposure of the gastric mucosa to a plethora of carcinogens present in betel and tobacco may provide a favourable milieu for neoplastic transformation. In light of the unique aetiological predisposition, it is worthwhile to investigate the prognostic profile of ESCC in the Indian population.

In a comprehensive analysis of molecular prognostic factors, Shimada and colleagues [4] reported that cyclin D1, E-cadherin, epidermal growth factor receptor (EGFR), pN, gender and cell growth capability are important predictors of patient survival and recurrence in the Japanese population. However, the relevance of the pRb protein, the central cog of the cell cycle regulation machinery as a molecular marker remains to be investigated. p53, MDM2, p21, p16, pRb and cyclin D1 constitute the most important proteins orchestrating the cell cycle regulation. To elucidate the prognostic significance of various cell cycle regulators, we examined the expression of six putative molecular markers: p53, MDM2, p21, p16, pRb and cyclin D1 by immunohistochemistry. The data were subjected to multivariate logistic regression analysis and the Kaplan–Meier method to identify predictors of prognosis for various clinicopathological parameters related to tumour progression.

Section snippets

Tumour specimens

Tumour and matched normal oesophageal tissue specimens from a distal site were collected from 100 ESCC patients who underwent curative oesophagectomy in the Surgical Oncology Unit of the Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India, with the prior consent of the patients. Tissue samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis. The clinical and pathological data were recorded in a predesigned proforma

Alterations in the p53-MDM2-p21 pathway in human oesophageal squamous cell carcinoma

The results of the immunohistochemical analysis of the cell cycle regulatory proteins, p53, mdm2, p21, p16, pRb and cyclin D1 in 100 ESCC patients in relation to clinicopathological parameters are summarised in Table 1. The photomicrographs in Fig. 1 illustrate representative immunostaining patterns for p53, MDM2 and p21 proteins in normal oesophageal tissues (Fig. 1a, c and e, respectively). In ESCCs, p53 protein was localised predominantly in the nuclei of tumour cells (Fig. 1b).

Discussion

To the best of our knowledge, this is the first composite report demonstrating frequent alterations in the expression of major cell cycle regulatory proteins of the p53-MDM2-p21 and p16-pRb-cyclin D1 pathways and their association with various clinicopathological parameters of oesophageal squamous cell carcinoma and disease prognosis in the Indian subcontinent. Mutations in both of these regulatory pathways have been reported in several human tumours [18]. Interestingly, all the ESCC cases

Acknowledgements

The contract grant sponsor was the Council of Scientific and Industrial Research, India.

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A number of studies have indicated that overexpression of MDM2 in tumors correlates with a poor prognosis for cancer patients, especially in breast cancer [7,8]. In addition, studies have indicated that higher MDM2 expression often occurs in metastatic and recurrent cancers than in primary tumors [9]. MDM2 gained increasing attention following its identification as the oncogene that negatively regulates the tumor suppressor p53.
2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ) is a novel synthesized combretastatin A-4 (CA-4) analog that can be classified as a microtubule inhibitor. Our previous study demonstrated that SQ induced G2/M phase arrest and promoted apoptosis progression in breast cancer cells. In the present study, we found that SQ dissociated the MDM2-p53 complex and directly induced MDM2 degradation through the ubiquitin-dependent proteasome pathway in MCF-7 and MDA-MB-231 cells. Further, p53 was activated by SQ through regulation of its transcription, translation, and post-translation modification. More specifically, we demonstrated that SQ induced caspase-dependent but p53-independent apoptosis, and this apoptosis is associated with the inhibition of MDM2. We also showed that SQ exhibited superior in vivo efficacy and low toxicity than CA-4. The immunofluorescence histochemistry study indicated that SQ also inhibited MDM2 expression in vivo. In summary, we report for the first time that SQ shows excellent anti-breast cancer activity in vivo and in vitro and induces p53-independent apoptosis, which is associated with MDM2 inhibition. Therefore, the novel compound SQ has potential for therapeutic treatment of both wild-type and mutant p53 breast cancer.
Transcription factor OCT4 promotes cell cycle progression by regulating CCND1 expression in esophageal carcinoma
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CCND1 expression was associated with the postoperative disease-free survival and overall survival of the patients. Therefore, CCND1 expression can be used as a prognostic indicator for esophageal cancer patients [24]. In summary, the disorder of cell cycle regulation is one of the important molecular events in the development of esophageal cancer, which causes cancer cells to have more proliferative and metastatic capacity.
The CCND1 gene is overexpressed in esophageal cancer and accelerates cell cycle progression. However, the mechanism whereby the upstream genes or factors directly regulate CCND1 expression remains unknown. By analyzing the 5′-UTR region of the CCND1 gene, we found that this region contains an octamer motif (ATTTTGCAT), which suggests that the expression of CCND1 might be directly associated with octamer-binding transcription factor 4 (OCT4). In this study, the wild-type and the octamer motif-mutanted CCND1 promoters were cloned, and their corresponding luciferase reporter vectors were then constructed to study the molecular mechanism by which OCT4 regulates the expression of CCND1 and influences the biological behaviors of esophageal cancer cells. The results indicated that suppressing the expression of CCND1 and OCT4 in esophageal cancer cells reduced cell proliferative and invasive abilities, induced cell cycle G1-phase arrest, and slowed the growth of xenografts in nude mice. Suppression of OCT4 expression significantly decreased the wild-type CCND1 promoter activity and down-regulated the expression of CCND1, but did not affect the activity of the mutant promoter. Whereas, suppression of CCND1 did not affect OCT4 expression, suggesting that OCT4 regulates CCND1 expression by activating the CCND1 promoter and subsequently promoting cell cycle progression. The results revealed and confirmed that OCT4 is the upstream factor that directly binds to the CCND1 promoter to regulate CCND1 expression, then to promote cell cycle progression and accelerate the proliferation and invasion of esophageal cancer cells. This finding may significantly contribute to elucidating the regulatory mechanism involved in the cell cycle progression of esophageal cancer cells and may aid in screening potential gene targets for the biological therapy of esophageal cancer.
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MDM2 is a RING finger-containing E3 enzyme involved in eukaryotic protein degradation via the ubiquitin proteasome system. Overexpression of the human homologue of MDM2, referred to as HDM2, occurs in diverse human malignancies (13, 14). Thus, MDM2 expression appears to correlate with an increased risk of distant metastases, which may contribute to an overall poorer prognosis for patients with tumors that overexpress MDM2 (15).
The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor.
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Alterations in p53 and pRb pathways and their prognostic significance in oesophageal cancer

Abstract

Introduction

Section snippets

Tumour specimens

Alterations in the p53-MDM2-p21 pathway in human oesophageal squamous cell carcinoma

Discussion

Acknowledgements

Eur. J. Cancer Oral Oncol.

American Journal of Pathology

Cell

Mol. Cell.

Ann. Thorac. Surg.

Guidelines for handling oesophageal biopsies and resected specimens and the reporting

J. Clin. Pathol.

Oesophageal squamous cell carcinomapathology and prognosis

World J. Surg.

Prognostic factors of oesophageal squamous cell carcinoma from the perspective of molecular biology

Br. J. Cancer

High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines

Cancer Res.

Amplification and expression of the human cyclin D gene in oesophageal cancer

Cancer Res.

p53 tumour supressor gene mutation in early oesophageal precancerous lesions and carcinoma among the high risk populations in Henan

China Cancer Res.

Oesophagus

High prevelance of p53 gene alterations and protein overexpression in human oesophageal cancercorrelation with dietary risk factors in India

Clin. Cancer Res.

Ethnic variations in the occurrence of gastrooesophageal cancers

Clin. Gastroenterol.

p53 overexpression in laryngeal squamous cell carcinoma and sysplasia

J. Clin. Pathol.

Altered p16/MTS1/CDKN2 and cyclin D1/PRAD-1 gene expression is associated with the prognosis of squamous cell carcinoma of the esophagus

Clin. Cancer Res.