TROP2 is epigenetically inactivated and modulates IGF-1R signalling in lung adenocarcinoma

Abstract

Trop-2, a cell surface glycoprotein, contains both extracellular epidermal growth factor-like and thyroglobulin type-1 repeat domains. Low TROP2 expression was observed in lung adenocarcinoma tissues as compared with their normal counterparts. The lack of expression could be due to either the loss of heterozygosity (LOH) or hypermethylation of the CpG island DNA of TROP2 upstream promoter region as confirmed by bisulphite sequencing and methylation-specific (MS) polymerase chain reaction (PCR). 5-Aza-2'-deoxycytidine treatment on lung cancer cell (CL) lines, CL1-5 and A549, reversed the hypermethylation status and elevated both TROP2 mRNA and protein expression levels. Enforced expression of TROP2 in the lung CL line H1299 reduced AKT as well as ERK activation and suppressed cell proliferation and colony formation. Conversely, silencing TROP2 with shRNA transfection in the less efficiently tumour-forming cell line H322M enhanced AKT activation and increased tumour growth. Trop-2 could attenuate IGF-1R signalling-mediated AKT/β-catenin and ERK activation through a direct binding of IGF1. In conclusion, inactivation of TROP2 due to LOH or by DNA methylation may play an important role in lung cancer tumourigenicity through losing its suppressive effect on IGF-1R signalling and tumour growth.

Figure 1. LOH and downregulation of TROP2 expression in lung adenocarcinomas

1. TROP2 is located on chromosome 1p32-p31, between markers D1S3721 and D1S209, which show frequent LOH in lung cancer samples. AD, adenocarcinoma; SQ, squamous cell carcinoma; NSCLC, non-small cell lung cancer.

2. The expression patterns of TROP2 in normal and cancer tissues, as modified from the ECgene database (http://genome.ewha.ac.kr/ECgene). The black arrow indicates the expression level of TROP2 in normal lung tissue. The expression of TROP2 in human NBE cells and lung CL were confirmed by RT-PCR.

3. The expression levels of TROP2 in various lung CL lines were measured by RT-PCR.

4. The protein levels of Trop-2 were determined by Western blot analysis.

Figure 2. Downregulation of TROP2 is associated with DNA hypermethylation of the TROP2 promoter region in lung CL lines

1. The putative promoter region of TROP2 was predicted using the promoter scan website (http://www-bimas.cit.nih.gov/molbio/proscan). The transcription and translation start sites are marked as ▾ and ▿, respectively. The numbers above the sequence indicate the CpGs that are predicted to be methylation sites. The boxed sequence (GGGCGG) is a putative Sp1 binding site. The underline ‘→’ and ‘←’ indicate the designed forward and reverse primers site on promoter region for MS-PCR analysis.

2. The methylation status of the putative promoter region in various cell lines was examined by bisulphite sequencing analysis. Each spot indicates one methylation site (CpG). The forward primer contains 29th and 30th CpG sites. The reverse primer contains 50th, 51st and 52nd CpG sites.

3. MS-PCR analysis of methylation in the various lung cancer lines by using specific methyl- and unmethyl- primer sets.

4. Treatment of cells with 5-aza-2′-DC dose-dependently restored TROP2 expression in A549 and CL1-0 cells. M and U indicated amplified by using methyl- and unmethyl- detecting primers. Panels 3 and 4 were detected by RT-PCR and panels 5 and 6 were measured by Western blot.

Figure 3. Epigenetic downregulation of TROP2 in lung cancer tissues

1. The methylation patterns in a number of lung cancer and normal tissue pairs were determined by MS-PCR.

2. In nearly 60% (10/16) of the pairs, the cancer tissues showed promoter region hypermethylation compared to the normal tissues.

3. Trop-2 protein expression in a number of paired samples was assessed by Western blot analysis. The densitometry was analysed by TL100 Gel Analysis Software (Nonlinear Dynamics Ltd, UK).

4. Immunohistochemical analysis of paired tissues (normal bronchial epithelium vs. adenocarcinoma) from two lung adenocarcinoma patients. In patient No. 1, the cancer tissue and normal bronchial epithelium showed nearly equal amounts of Trop-2. In patient No. 2, the NBE cells showed TROP2 expression, but the cancer tissue did not.

Figure 4. TROP2 expression modulates cell proliferation and colony formation

1. Trop-2 was overexpressed in H1299 cells and knocked down in H322M cells, and the activities of AKT and ERK were determined by Western blotting with anti-pAKT and anti-p-ERK antibodies, respectively.

2. Forced expression of Trop-2 in H1299 cells inhibited cell proliferation compared to the vector control, as determined by cell counting. Conversely, Trop-2 knockdown by either lenti-shTROP2A or lenti-shTROP2C enhanced cell proliferation.

3. The tumour growth ability of lung CL was measured by the formation of colonies in soft agar. The results are presented as means ± SD, data were compared between groups using the t-test, and p < 0.05 was considered statistically significant when compared to the control.

Figure 5. TROP-2 knockdown promotes xenograft tumour growth in mice

1. H322M cells were infected with lenti-shTROP2A or lenti-shLacZ (control) and injected into nude mice, and tumour volumes were monitored for 6 weeks. The results are presented as means±SEM, data were compared between groups using the t-test, and p < 0.05 was considered statistically significant when compared to the control.

2. Photos of tumour-bearing mice, with tumours indicated by black arrows.

3. H&E staining of tumour tissues obtained from mice inoculated with lenti-shTROP2A or lenti-shLacZ.

Figure 6. Trop-2 can bind to IGF-1 and interfere with IGF-1R signalling

1. BLAST analysis of Trop-2 against the NCBI database (http://www.ncbi.nlm.nih.gov/) shows that the members of the TROP and insulin growth factor binding protein (IGFBP) families share a conserved thyroglobulin type-1 domain. Black and purple boxes indicated the glycosylation and phosphorylation sites, respectively.

2. Vectors encoding TROP2 and mIGF-1 were co-transfected into H1299 lung CL, and the interaction of TROP2 and mIGF-1 was examined by co-IP. WB, western blot.

3. Overexpression of TROP2 in H1299 cells reduced the activation of IGF-1R signalling, while TROP2 knockdown activated IGF-1R signalling in H322M cells.

4. Forced expression of TROP2 in H1299 cells suppressed the IGF-1-induced phosphorylation of AKT.

5. The expression of downstream mediators of IGF-1R signalling (e.g. β-catenin) decreased when TROP2 was overexpressed in H1299 and H23 cells, and increased following TROP2 knockdown in H322M and PC-9 cells. Slug expression was induced by TROP2 knockdown.

Figure 7. The proposed role of TROP2 in lung tumourigenesis

When TROP2 is silenced by DNA methylation, Trop-2-mediated suppression of IGF-1R signalling decreases. This may lead to cancer progression (e.g. invasion, metastasis and/or angiogenesis) via activation of IGF-1R signalling and its downstream mediators β-catenin and slug.