Pathogenesis sequences in Gejiu miners with lung cancer: an introduction

Abstract

Tin miners in Gejiu, Yunnan Province, China are at high risk of developing lung cancer with significant occupational characteristics. Tissue samples from these miners presented pathological characteristics, such as fibroplasia in carcinomas, peri-cancerous tissue in lung cancers, and hyperplasia and dysplasia of epithelial cells in peri-cancerous tissue. Carcinomas induced by Yunnan tin mine dust in the animal experiment underwent inflammation, fibroplasia, hyperplasia, dysplasia, and carcinogenesis of epithelial cells. A correlated and synergistic relationship was observed between bronchial epithelial cell transformation and fibroblast activation in vitro induced by mine dust. Fibroblast hyperplasia and activation are important factors that promote the transformation and carcinogenesis of epithelial cells. Our findings suggested that pulmonary fibrosis may increase the risk and promote the occurrence of lung cancer, which can lead to lung fiber hyperplasia.

Keywords

Yunnan tin mine / miners / lung cancer

Introduction

Lung cancer is the most common malignant tumor and remains the leading cause of cancer deaths worldwide. As epidemiological studies showed that tin miners in Gejiu, Yunnan Province, China are at high risk of developing lung cancer, this population has attracted considerable attention worldwide because of the high incidence and mortality of this cancer. The incidence rate of lung cancer among all Yunnan tin miners reached 491.96/100 000 in 2000-2004 [ 1]. The high incidence of lung cancer manifests significant occupational characteristics. For example, the incidence rate of lung cancer was 790.3/100 000 among tin miners who worked underground and 39.03/100 000 in the general population [ 1]. The mortality of lung cancer among tin miners was 14 times higher than that of the general population [ 1].

A large-scale etiology study of lung cancer among Gejiu miners was conducted in the 1970s and 1980s. The results showed that the pathogenesis of lung cancer is related to the synergistic effect between metallic (e.g., radon and its derivatives, arsenic, lead, and iron) and non-metallic elements in tin mine dust [ 2]. As the pathogenesis of lung cancer in Gejiu tin miners remains partially understood, we conducted a series of studies in the past two decades to elucidate the pathogenesis. Our findings are described in this report.

Materials and methods

Human samples

Twenty samples of lung cancer tissue from miners, 20 samples of lung cancer tissue from non-miners, and 10 samples of normal lung tissue were collected for hematoxylin-eosin (H&E) stain, in situ hybridization, and immunohistochemistry. The following antibodies were used in this study: anti-P53 (Zymed, US), anti-P21Ras (Maxim Biotech, US), anti-HnRNP (Santa Cruz, US), anti-DAPK (BD, US), anti-MDM2 (Zymed, US), anti-SP-B (Maxim Biotech, US), anti-TGF-β1 (Maxim Biotech, US), anti-FHIT (Zymed, US), and anti-SMA (DAKO).

Animal model

A total of 190 F344 rats (4-6 weeks age, SPF/UAF; purchased from the Beijing Weitonglihua Experimental Animal Technical Company) were randomly divided into four groups for lung cancer induction: mine dust perfusion group (100 rats) and three control groups, namely, 2% furfural group (30 rats), saline infusion group (30 rats), and normal control group (30 rats). After 1 week of perfusion, one rat was sacrificed in the mine dust perfusion group every week until all rats were sacrificed (100 weeks), whereas one rat was terminated every month in the other three groups until all rats were sacrificed.

Cell experiment

The immortalized human bronchial epithelial cell line BEAS-2B (ATCC, US) and human embryo lung fibroblast cell line WI-38 (ATCC, US) were cultured in MEM containing 5% and 10% FBS, respectively, at 37 °C and 5% CO₂ with saturated humidity. All cells were exposed to mine dust from the first generation up to the ninth generation. The two cell lines were then co-cultured from the 11th generation to the 40th generation. Concanavalin A (ConA) agglutination assay and anchorage-independent growth assay were used for testing the transformation of epithelial cells. α-SMA was used to assess fibroblast activation.

Results and discussion

Lung cancer tissue sections of tin miners

We microscopically examined a large number of lung cancer tissue from Gejiu miners and found 27 cases of squamous cell carcinoma from 41 lung cancer samples. The major histologic type of lung cancer in tin miners is squamous cell carcinoma with clear stromagenesis and diffuse pulmonary fibrosis (27/41), followed by adenocarcinoma, adenosquamous carcinoma, and several other types [ 3]. Bronchial epithelial hyperplasia, squamous metaplasia, bronchial epithelial dysplasia, and carcinoma in situ were also found next to carcinomas. The characteristic pathological changes are the large amount of mine dust deposition in the cancerous and surrounding tissue, clear stromagenesis, diffuse pulmonary fibrosis, and thickening of alveolar septa fiber. Alveolar type II epithelial hyperplasia, dysplasia, and even transition were present next to squamous carcinomas (Figs. 1 and 2). We also detected two cases of squamous metaplasia derived from alveolar epithelia. These findings suggested that squamous metaplasia not only occurs in the bronchial epithelia, but also in the alveolar epithelia; hence, one-third of squamous carcinoma in clinical samples is peripheral lung cancer [ 4].

The expression of P53, FHIT, N-ras, c-myc/c-fos, TGFβ1, DAPK, and SP-B in lung cancer tissue statistically significantly differed between tin miners and non-miners [ 3- 9]. We previously reported that P53 mutation, deletion, and abnormal FHIT expression occur in tin miners with lung cancer, particularly in hyperplastic epithelial cells of the bronchial and alveolar epithelia next to carcinoma. Therefore, the expression of P53 and FHIT can be an early molecular event in the development of lung cancer induced by Yunnan tin mine dust [ 4- 6]. TGFβ1, a key factor that leads to pulmonary fibrosis, was overexpressed in 63.96% of miners with lung cancer and in 34.07% of tin miners. These results indicated that TGFβ1 may play an important role in the high incidence rate of lung cancer among miners [ 9]. We also previously reported that the hyperplasia of alveolar type II epithelia may be related to pulmonary fibrosis, and a small number of squamous cancer expressed SP-B [ 3].

Basing on these findings, we conducted in vitro and in vivo experiments to address the following questions: can lung squamous carcinomas originate from the hyperplasia and dysplasia of alveolar type II epithelia in tin miners; what is the actual relationship among diffuse pulmonary fibrosis, lung epithelial hyperplasia, and canceration in tin miners with lung cancer; is pulmonary fibrosis more likely to cause the tumorigenesis or is canceration more likely to induce interstitial hyperplasia during mine dust-induced tumorigenesis; does a deeper connection between pulmonary fibrosis and lung cancer exist; and what is the molecular pathology mechanism of miners with lung cancer?

Animal carcinogenesis experiments

We established an experimental animal model of lung cancer with mine dust inhalation in F344 rats [ 10] to study the pathological and dynamic changes of related proteins during cancer development. We also examined changes in the early lung morphology and gene expression in dust-exposed rats and compared these changes with the results from tin miners with lung cancer.

A consistent histological finding in the animal models is the initial induction of inflammation and respiratory cell injury, followed by epithelial proliferation and stromal reaction and then metaplasia, particularly squamous metaplasia. The hyperplasia of fibrous tissue and local fibrosis were apparent in areas deposited with mine dust. Finally, dysplasia and carcinoma formation occurred [ 10] (Figs. 3-6). In addition, squamous metaplasia was observed not only in the bronchial epithelia, but also in the alveolar epithelia (Fig. 5). The squamous metaplasia of the alveolar epithelia underwent dysplasia and formed squamous cell carcinomas. Furthermore, the hyperplasia of ATII can transform into atypical adenomatous hyperplasia and even adenocarcinoma in animal models [ 10]. Collagen deposition and diffuse pulmonary fibrosis were further observed next to carcinoma and in the area of mine dust deposition, in which the activated fibroblasts expressed α-SMA. Our experiments confirmed that pulmonary fibrosis occurred before cancer formation. Fibrosis formation is also likely to contribute to the accumulation of material that induces cancer, thereby increasing the risk of lung cancer [ 10]. We speculated that a similar sequence of events can likely occur among miners with lung cancer. Thus, the established animal model with mine dust inhalation can be used to dynamically observe the relationship between pulmonary fibrosis and lung cancer. The relationship between pulmonary fibrosis and lung carcinogenesis caused by mine dust inhalation must be further investigated. As the overexpression of MDM2, P21^ras, P53, HnRNP, and FHIT protein was detected in F344 rats in the early stage of lung cancer induced by Yunnan tin mine dust [ 11], similar changes can likely occur in miners with lung cancer. These molecular events occur before morphological changes.

In vitro experiments

We used the immortalized bronchial epithelial cell line BEAS-2B (ATCC, US) and human embryo lung fibroblast cell line WI-38 (ATCC, US) to establish an in vitro co-culture model of epithelial cells and fibroblasts [ 12]. The model was used to simulate the interaction of epithelial-stromal cells in vivo and investigate the mechanism and interaction between lung epithelial cell transformation and fibroblast activation induced by Yunnan tin mine dust. Both cell lines were separately exposed to mine dust from the first generation to the ninth generation and then co-cultured from the 11th generation (1:1 ratio to start with) to the 40th generation. We found that Yunnan tin mine dust can induce epithelial cell transformation, as determined by ConA agglutination and anchorage-independent growth assays (Figs. 7 and 8), and fibroblast activation, as determined by the expression of α-SMA (Fig. 9), a specific marker of fibroblast activation in the in vitro co-culture model [ 12]. Furthermore, these results suggested that tin mine dust can induce epithelial cell transformation and fibroblast activation without the co-culture. Furthermore, tin mine dust-activated fibroblasts can promote the proliferation and transformation of epithelial cells. The activation of fibroblasts can be similarly promoted by dust-exposed epithelial cells in vitro. However, the dust-exposed fibroblasts were insufficient to induce the transformation of epithelial cells that were not exposed to tin mine dust as previously reported [ 13]. We previously reported that Gejiu mine dust is a non-genotoxic carcinogen as determined through single-cell gel electrophoresis (comet assay) and chromosome aberration test [ 14]. These results indicated that carcinogenic factors in tin mine dust can induce epithelial cell malignant transformation, and lung epithelial cells are the main target cells during dust-induced tumorigenesis [ 13].

The aberrant expression of P53, MDM2, P21, P16, and FHIT was observed in the transformed epithelial cells in the in vitro co-culture model [ 15, 16]. The P53 gene abnormality can be one of the key steps in the early malignant transformation of epithelial cells induced by tin mine dust [ 15]. Furthermore, FHIT can be a Yunnan tin mine dust target that induces lung cancer and the downregualtion or loss of its protein expression can be a valuable biological marker to predict the risk of lung cancer [ 16].

In addition to different carcinogens, mine dust contains SiO₂, which causes pulmonary fibrosis. Therefore, Yunnan tin mine dust is a suitable material that can be used to study the correlation among lung fibrosis, fibroblast activation, and lung cancer.

Conclusions

Epithelial cell transformation and fibroblast activation exhibit a correlated and synergistic relationship during carcinogenesis induced by Gejiu tin mine dust. Our findings suggested that pulmonary fibrosis can increase the risk and promote the occurrence of lung cancer, which can lead to lung fiber hyperplasia. We proposed a schematic for the induction of lung epithelial cell transformation and fibroblast activation by Yunnan tin mine dust and their associated interaction (Fig. 10). The interaction between epithelial cell transformation and fibroblast activation involves multiple signaling pathways. Investigation of the downstream molecular events of these signaling pathways is currently ongoing.

References

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