Decreased PITX1 homeobox gene expression in human lung cancer

Summary

The PITX1 (pituitary homeobox 1) gene has essential roles in human development and has been considered a tumor suppressor in various cancers. However, in lung cancer the role of PITX1 remains to be elucidated. In this study, we analyzed the expression of PITX1 at both mRNA and protein levels in human lung cancer. The reduced PITX1 expression was found in cancer cell lines test compared to normal human bronchial epithelia cells (HEBC) and small airway epithelia cells (SAEC) by Northern blot analysis and RT-PCR as well as Western blot analysis. In primary lung tissues, PITX1 mRNA was found to be downregulated in the majority of tumors compared with normal lung tissues. An association between the lack of PITX1 mRNA expression and higher tumor grade was observed. A tissue microarray containing 135 primary lung carcinomas was analyzed by immunohistochemistry. Eighty-four cases (62%) exhibited no expression of PITX1 and the lower expression of PITX1 was significantly linked to higher tumor stages. Additionally, PITX1 was found to be upregulated in lung cancer cell lines H2228 and H526 after they were exposed to a differentiation modifying agent 5-bromodeoxyuridine (BrdU). Since homeobox genes are known to transcriptionally regulate key cellular processes and associated with differentiation and carcinogenesis, we suggest that PITX1 might be linked to lung cancer development and progression.

Introduction

Lung carcinoma is the most fatal cancer worldwide, alone in the United States, the estimated number of deaths due to lung cancer will exceed 100,000 [1]. Despite the major progress made in cancer treatment during the last decades, currently still more than 90% of all cases of lung cancer diagnosed in Europe die within 5 years, a proportion that compares poorly with the mortality of breast or colon cancer [2]. To achieve new therapeutic approaches for this fatal disease, a better understanding of the molecular mechanisms underlying the complex process of tumorigenesis in lung cancer is largely required.

Cancer is considered a genetic disease, because tumor growth is due to functional imbalance between oncogenes and tumor suppressor genes, and because genetic alterations, at either the chromosomal or molecular levels, have been identified in most cancers including lung cancer [3], [4]. Over the past two decades, a number of oncogenes and tumor suppressor genes have been identified, providing new insights into human oncogenesis as well as into the growth, differentiation, and maintenance of the normal cell population.

In order to identify new candidate genes in lung carcinogenesis, we previously performed suppression subtractive hybridization (SSH) to reveal lung cancer associated genes, in addition to expression profiling by using a 24,000 element cDNA microarrays [5], [6], [7]. Comparing the gene expression between normal human bronchial epithelial cells (HBEC) and lung squamous carcinoma cells, we cloned two cDNA libraries representing mainly the genes that are over-expressed and under-expressed in HBEC and tumor cell line, respectively. The clone HBECII-79 with high similarity to the human homeodomain pituitary transcription factor PITX1 gene was found in the library enriched for the genes, which were downregulated in tumor cells. As little is known about possible tumor-inhibitory roles of PITX1 in lung cancer, we focused on PITX1 expression in lung cancer.

Pituitary homeobox 1 (PITX1/PTX1), also called Backfoot (BFT), is a bicoid-related homeobox transcription factor that is involved in transcription of the pro-opiomelanocortin (POMC) gene [8], [9]. PITX1 plays a role in differentiation and formation of pituitary cells as well as in development of oral epithelium, first branchial arch, and its derivatives [10]. Pitx1 is expressed throughout the developing hindlimb but not forelimb bud. Misexpression of Pitx1 in the chick wing buds causes them to developed into limbs with some morphological characteristics of hindlimbs [11], [12]. In lung, the role of Pitx1 has not been well elucidated, however, the Pitx2 gene, which is highly homologous to Pitx1 is required for left–right asymmetry of the lungs but not for other organs, indicating an important role of the pituitary homeobox gene in lung development [13].

In this study, we evaluated the expression of PITX1 in lung cancer cell lines and primary lung tumors. We demonstrated the downregulation of PITX1 at both mRNA and protein levels in lung cancer cell lines. Additionally, lower expression of PITX1 protein was found to be significantly linked to tumors with higher stages.