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FRMD3, a novel putative tumour suppressor in NSCLC

Abstract

Lung cancer including non-small cell lung carcinoma (NSCLC) represents a leading cause of cancer death in Western countries. Yet, understanding its pathobiology to improve early diagnosis and therapeutic strategies is still a major challenge of today's biomedical research. We analyzed a set of differentially regulated genes that were identified in skin cancer by a comprehensive microarray study, for their expression in NSCLC. We found that ferm domain containing protein 3 (FRMD3), a member of the protein 4.1 superfamily, is expressed in normal lung tissue but silenced in 54 out of 58 independent primary NSCLC tumours compared to patient-matched normal lung tissue. FRMD3 overexpression in different epithelial cell lines resulted in a decreased clonogenicity as measured by colony formation assay. Although cell attachment capabilities and cell proliferation rate remained unchanged, this phenotype was most likely owing to induced apoptosis. Our data identify FRMD3 as a novel putative tumour suppressor gene suggesting an important role in the origin and progression of lung cancer.

Main

Lung cancer, with its two major subtypes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), is a leading cause of cancer-related mortality worldwide. Hitherto, therapy for NSCLC, which accounts for about 80% of lung cancer cases (Greenlee et al., 2000), is still generic and largely ineffective, and thus patients’ prognosis with advanced lung cancer remains poor (Shaw et al., 2005). A more detailed knowledge on the molecular principles of lung cancer development may contribute to a meaningful therapeutic progress. Particularly, identification and functional characterization of genes with altered expression in tumours represents a promising approach to unravel crucial processes involved in NSCLC. Concomitantly, molecular studies have revealed several genetic aberrations in lung cancer pathogenesis accompanied by altered expression and function of oncogenes, such as c-myc, K-ras, Raf and Bcl-2 or loss of tumour suppressor gene expression and function, such as p53, Rb and BRCA1-binding protein BAP-1 (reviewed by Forgacs et al., 2001; Minna et al., 2002; Rapp et al., 2003).

Applying gene expression analysis on samples of chemically induced tumours of mouse back skin, we have identified a comprehensive list of differentially regulated genes in epidermal squamous cell carcinoma (Breitenbach et al., 2001; Schlingemann et al., 2003; Hummerich et al., 2006). In order to search for novel diagnostic lung cancer biomarkers and potential therapeutic targets, we measured the expression levels of a set of genes identified by this approach in specimens of NSCLC patients. When we analysed a series of 27 pulmonary squamous cell carcinomas (pSCC) and 21 adenocarcinomas (AC) and the corresponding normal unaffected lung tissue (Figure 1a and Supplementary Figure 1), transcripts of ferm domain containing protein 3 (FRMD3) (alias protein 4.1O), a member of the protein 4.1 superfamily, were detected in normal lung tissue samples of all patients (N). Interestingly, in tumour tissue (T) FRMD3 transcript levels were significantly reduced. Moreover, hybridization of a cancer profiling array including a series of 10 patient-matched normal and tumour tissue samples comprising five pSCC, two AC and three not further assigned lung tumours with a human FRMD3 specific probe revealed impaired expression levels in nine out of 10 NSCLC samples (Figure 1b and Supplementary Figure 2). In total, 54 out of 58 NSCLC patients exhibited a reduction in FRMD3 transcription in tumour tissue (Supplementary Table 1b) supporting a correlation between NSCLC and FRMD3 loss. However, reduced FRMD3 transcription could not be correlated to one specific tumour stage (Supplementary Table 1), suggesting that it occurs early during lung cancer development. Taken together, these data indicate that NSCLC is highly associated with diminished FRMD3 transcription.

Figure 1
figure1

FRMD3 and DAL-1 transcript levels in normal tissue, primary tumours and tumour cell lines. (a) Expression of FRMD3 and DAL-1 in a series of patient-matched NSCLC tumours (T, detailed data of tumours’ pathology are listed in Supplementary Table 1a) and normal (N) lung tissue samples were analysed by sqRT–PCR (method: see Supplementary Figure 1). Plasmid=cDNA of FRMD3 or DAL-1; +=lung cDNA positive for FRMD3 and DAL-1 expression (b) Expression of FRMD3 and DAL-1 transcripts in lung, rectum, kidney and skin cancer (T), normal tissue (N) and different tumour cell lines was analysed by hybridization of a cancer profiling array (BD Clontech, Heidelberg, Germany) using the corresponding cDNA probes.

FRMD3 is of particular interest as members of the 4.1 protein superfamily, which share the highly conserved membrane-association domain FERM, have already been identified as tumour suppressors (reviewed by Sun et al., 2002). The most prominent protein 4.1 tumour suppressors are Merlin/NF2/Schwannomin (reviewed by Sherman and Gutmann, 2001), protein 4.1R (Huang et al., 2001; Robb et al., 2003) and protein 4.1B (Tran et al., 1999; Gutmann et al., 2000; Perry et al., 2000; Singh et al., 2004; Kuns et al., 2005; Gerber et al., 2006). The minimal growth inhibitory domain of protein 4.1B DAL-1 (differentially expressed in adenocarcinoma of the lung) and closest relative to FRMD3 with 38% identity on protein level (Ni et al., 2003) is frequently lost or reduced in NSCLC and in meningiomas (Tran et al., 1999; Gutmann et al., 2000, 2001; Perry et al., 2000). Moreover, re-introduction of DAL-1 into NSCLC, breast carcinoma and meningioma cell lines results in growth suppression due to cell-cycle arrest and, at least in part, to induction of apoptosis (Tran et al., 1999; Charboneau et al., 2002; Kuns et al., 2005; Robb et al., 2005; Gerber et al., 2006). When we measured DAL-1 mRNA levels on the entire tumour sample series analysed for FRMD3 transcription, DAL-1 downregulation was found in about half of the pSCC and AC samples examined (Figure 1 and Supplementary Figure 1). Thus, the correlation of lung cancer development and loss of FRMD3 expression is even stronger than the one described for the known tumour suppressor protein 4.1B/DAL-1 (Tran et al., 1999).

Downregulation of protein 4.1B/DAL-1 transcription has been reported not only in NSCLC but also in other tumours (Tran et al., 1999; Gutmann et al., 2000, 2001; Perry et al., 2000). To determine whether reduced FRMD3 expression can also be associated with other human epithelial malignancies, we hybridized a cancer profiling array with FRMD3 and DAL-1-specific cDNA probes and compared different patient-matched pairs of normal and tumour tissue samples such as rectum, kidney and skin cancer. Whereas DAL-1 expression was significantly downregulated in kidney cancer (8/10), no significant correlation between cancer development and loss of FRMD3 transcription was obvious in other tumour types examined (Figure 1b). This strongly suggests that loss of FRMD3 transcription is restricted to lung cancer.

To investigate whether FRMD3 shares a similar suppressive function on cell growth or survival as DAL-1 (Gutmann et al., 2001; Charboneau et al., 2002; Kuns et al., 2005; Robb et al., 2005; Gerber et al., 2006), we performed colony formation assay (CFA) with transiently transfected HEK293, HeLa and A549 cells that show only marginal endogenous FRMD3 expression as measured by semiquantitative reverse transcription–polymerase chain reaction (sqRT–PCR) (Figure 2a) and hybridization of the cancer profiling array (Figure 1b). Ectopic FRMD3 expression, as proven by sqRT–PCR and Western blot (Figure 2b, left panel) reduced clonogenicity of HEK293 cells to approximately 32% compared to mock controls (Figure 2b, middle and right panels), which was not due to plating efficiency (data not shown). Decreased clonogenicity upon forced FRMD3 expression was verified in HeLa and A549 cells (Figure 2b, right panel), confirming that FRMD3 may act as a growth suppressor.

Figure 2
figure2

Clonogenicity, cell proliferation and cell cycle regulation in cells overexpressing FRMD3. (a) Expression of FRMD3 in different cell lines was determined by sqRT–PCR (method: see Supplementary Figure 1). As positive control cDNA of human adult ovary and unaffected (N) lung tissue was used. Tumour (T) tissue cDNA of the identical patient served as negative control. (b) HEK293, HeLa and A549 cells were transfected by calcium phosphate co-precipitation or following the Nucleofector procedure (Amaxa, Cologne, Germany) with either empty pcDNA3.1 vector (mock) or myc/his-tagged FRMD3. Ectopic FRMD3 expression (left panel) was monitored by RT–PCR (left panel, top; method: see Figure 1a) and by Western blot analysis (left panel, bottom): 30 μg protein lysate was resolved on a 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto Optitran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and probed with monoclonal α-myc (Invitrogen, Karlsruhe, Germany) and α-β actin antibodies (Sigma, Munich, Germany). β-Actin served as a control for equal quality and loading of cell extracts. For CFA, cells were transfected, plated on 6 cm dishes and grown for 6–10 days in the presence of Geneticin. To proof comparable transfection efficiency for all experiments, green fluorescent protein (GFP) expression was monitored by FACS analysis 24 h post transfection. Colonies were fixed, stained with crystal violet (middle panel) and counted. Colony numbers (means±s.e.m.) of three independent experiments are given (right panel). Mock control was set to 100%. (c) Cell cycle phases of mock and FRMD3-transfected HEK293 cells were analysed 72 h post transfection. After fixation in 70% ethanol and 2 h staining with 50 μg/ml propidium iodide (Sigma) cells residing in G0/G1, S or G2/M phases were visualized by FACS Calibur (BD, Heidelberg, Germany). One representative of three independent experiments is shown as histogram, percentage of cells in the different cell cycle phases were determined and means±s.e.m. of three independent experiments are reported.

To evaluate whether FRMD3 affects cell-cycle progression similar to DAL-1 (Kuns et al., 2005; Gerber et al., 2006), we performed fluorescence-activated cell sorting (FACS) analysis of propidium iodide stained mock and FRMD3 transfected HEK293 cells. No significant differences in the percentage of cells residing in G0/G1 (mock 54%, FRMD3 54%), S (mock 23%, FRMD3 24%) or G2/M phases (mock 23%, FRMD3 22%) were observed 24, 48 and 72 h after transfection (Figure 2c). In line with these data, we found no significant alteration in the percentage of BrdU-positive cells 48 and 72 h post transfection in mock versus FRMD3 or nontransfected HEK293 cells (Supplementary Figure 3 and data not shown). Similar data were obtained in control and FRMD3-expressing HeLa and A549 cells (data not shown). Taken together, these data suggest that FRMD3 overexpression has no impact on cell-cycle progression indicating that FRMD3 acts differently from protein 4.1/DAL-1.

Finally, we asked whether FRMD3-mediated reduction of clonogenicity is due to altered cell survival. Indeed, upon forced FRMD3 expression, we found significantly increased apoptosis of HeLa and HEK293 cells as measured by Annexin V staining. Compared to mock control specific apoptosis in FRMD3-expressing HeLa cells was twofold increased 30 (P=0.0057) (Figure 3a) and 50 h post FRMD3 transfection (data not shown). In HEK293 cells, the most prominent effect was detected at 72 h post transfection with a 4.7-fold increase in specific apoptosis (P=0.0067) (Figure 3a). Thus, FRMD3 overexpression efficiently induces apoptosis, which could explain the decrease in clonogenicity. JC-1 stain of transfected HEK293 cells 72 h post transfection revealed no significant impact of FRMD3 on the mitochondrial membrane potential (ΔΨ) (Figure 3b) excluding the induction of the intrinsic apoptotic pathway involving the mitochondria. However, clonogenicity of FRMD3-overexpressing HeLa cells was restored in the presence of caspase inhibitor (z-VAD-FKM) and raised from 35 to 82% (Figure 3c), indicating that active caspases are at least in part involved in FRMD3 action, which is similar to what has been reported for DAL-1-induced apoptosis (Jiang and Newsham, 2006).

Figure 3
figure3

Forced FRMD3 expression induces apoptosis via activation of caspases rather than alteration of mitochondrial membrane potential. (a) Rate of specific apoptosis: FRMD3 and mock transfected HeLa and HEK293 cells were cotransfected with CMV-GFP and stained with APC-coupled Annexin V (diluted 1:200; BD, Heidelberg, Germany) at the indicated time points post transfection. GFP positive cells were analysed by FACS Calibur (BD) for Annexin V positive staining. Rate of spontaneous apoptosis was deduced. Means±s.e.m. of three independent experiments are reported. (b) JC-1 stain (Molecular Probes, Karlsruhe, Germany) following the manufactures instructions was performed to determine cells with collapsed membrane potential (ΔΨ) in mock (34%) and FRMD3 (39%) (P=0.632) transfected HEK293 cells 72 h post transfection. Subsequent FACS Calibur (BD) analysis allowed the determination of the percentage of cells with collapsed ΔΨ. Means±s.e.m. of three independent experiments are reported, ns, not significant (c) CFA (method: see Figure 2b) was performed with mock and FRMD3-transfected HeLa cells in the absence (dimethylsulphoxide) or presence of 20 μ M caspase inhibitor z-VAD-FKM (Axxora, Lörrach, Germany). Inhibitor was added every 12 for 72 h. Colony numbers (means±s.e.m.) of four experiments are given. Mock control was set to 100%. mockDMSO/FRMD3DMSO: P=0.001; mockz−VAD−FKM/FRMD3z−VAD−FKM: P=0.034.

Recent studies assigned the FRMD3 protein to the protein 4.1 superfamily and reported focal expression for this gene in human adult ovary (Ni et al., 2003). Yet, information about its potential cellular or physiological function was missing. Our data identify FRMD3 as a novel putative tumour suppressor gene probably implicated in the origin and progression of lung cancer, further supported by its mapping to chromosome 9q which is frequently lost in NSCLC (Tran et al., 1999; Panani and Roussous, 2006). Loss of FRMD3 expression may be ultimately connected with a survival advantage of malignant cells but it remains to be determined whether FRMD3 might either interfere with the activity of transforming oncogenes, such as c-myc, K-ras, Raf and Bcl-2, or affect the activity of proteins associated with NSCLC and concomitantly known to play a crucial role in apoptosis such as p53, the death-associated protein kinase (DAPK) and the fragile histidine triad gene (FHIT) (reviewed by Forgacs et al., 2001; Panani and Roussous, 2006).

Functional studies with other 4.1 protein subfamily tumour suppressors, such as 4.1B/DAL-1 and 4.1R are at an advanced stage and provide comprehensive data on protein–protein interactions with other cellular factors, such as TSLC-1, 14-3-3, PRMT3 and hDlg, which might be critically involved in DAL-1 and 4.1R activity (Yageta et al., 2002; Hanada et al., 2003; Mao et al., 2003; Robb et al., 2004; Singh et al., 2004). On the basis of this knowledge, our future studies shall decipher the mechanism of FRMD3 action in apoptosis and elucidate the cause for loss of FRMD3 expression in NSCLC in the near future. Particularly, determining whether re-expression of FRMD3 will allow the elimination of malignant cells will open novel avenues for desperately needed therapeutic approaches in lung carcinogenesis.

Abbreviations

AC:

adenocarcinoma

CFA:

colony formation assay

DAL-1:

differentially expressed in carcinoma of the lung 1

FRMD3:

ferm domain containing protein 3

NSCLC:

non small cell lung carcinoma

pSCC:

pulmonary squamous cell carcinoma

sqRT–PCR:

semiquantitative RT–PCR

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Acknowledgements

We acknowledge Dr Marina Schorpp-Kistner for helpful discussion about the manuscript and associated work. We thank Dr Irena Crnković-Mertens for help with preparing the RNAs of some of the N/T-samples. NSCLC tissues were obtained according to the ethic votum 270/2001, version 2.0 (23 January 2005).

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Correspondence to B Hartenstein.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Haase, D., Meister, M., Muley, T. et al. FRMD3, a novel putative tumour suppressor in NSCLC. Oncogene 26, 4464–4468 (2007). https://doi.org/10.1038/sj.onc.1210225

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Keywords

  • protein 4.1
  • lung cancer
  • ferm containing 3
  • apoptosis

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