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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0b013e3181c6e330
Pathway of the Month

RBM5 as a Putative Tumor Suppressor Gene for Lung Cancer

Sutherland, Leslie C. PhD*†‡; Wang, Ke MD, PhD§; Robinson, Andrew G. MD∥

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*Tumour Biology Group, Regional Cancer Program of the Sudbury Regional Hospital; †Faculty of Medicine, Biomolecular Sciences Program, and Departments of Biology and Chemistry/Biochemistry, Laurentian University, Sudbury, Canada; ‡Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; §Department of Respiratory Medicine, The Second Affiliated Hospital of Jilin University, Changchun, Jilin, China; and ∥Medical Oncology, Regional Cancer Program, Sudbury Regional Hospital, Sudbury, Ontario, Canada.

Disclosure: The authors declare no conflicts of interest.

Address for correspondence: Leslie C. Sutherland, PhD, Tumour Biology Group, Department of Research, Regional Cancer Program of the Hôpital Régional de Sudbury Regional Hospital, 41 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 5J1. E-mail: lesutherland@hrsrh.on.ca

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Abstract

RBM5 is one member of a group of structurally related genes that includes RBM6 and RBM10. RBM10 maps to Xp11.23, and one allele is inactivated as a result of X chromosome inactivation. Both RBM5 and RBM6 map to 3p21.3, a tumor suppressor region that experiences loss of heterozygosity in the majority of lung cancers. Overexpression of RBM5, which encodes an RNA-binding protein involved in the regulation of alternative splicing and retards ascites associated tumor growth in immunocompromised mice, a phenomenon that may be related to an associated ability to modulate apoptosis. As part of our quest to gain a better understanding of how the proapoptotic activity of RBM5 might contribute to tumor suppressor function, we reviewed all the literature relating to RBM5 expression, with a focus on lung cancer. On the basis of the existing data, we suggest that—to more thoroughly assess the potential involvement of RBM5 as a lung cancer regulatory protein—more research is required regarding (a) the expression of not only full-length RBM5 but all of the alternate variants associated with the locus, in relation to histologic subtype and smoking history, and (b) the mutation status of various genes within the transforming growth factor-α signaling pathway, which may function to either directly or indirectly regulate RBM5 activity in RBM5-retaining lung cancers.

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Tumor Suppression Relating to 3p21.3

The earliest premalignant chromosomal aberration in human lung cancers is allele loss within the short arm of chromosome 3 at 3p21.3.1 This loss of heterozygosity occurs in practically all (>95%) small cell lung cancer (SCLC) tumors, the majority (>70%) of non-SCLC (NSCLC) tumors, and in the normal bronchial epithelium of some smokers and former smokers.2–4 The smallest lung cancer-specific deletion region (based on homozygous deletion studies relating to 3 established SCLC cell lines (GLC20, NCI-H740, and NCI-H1450) spans 370 kb, 19 genes (RBM6, RBM5, SEMA3F, GNAT1, NAT1/SLC38A3/G17, GNAI2, SEMA3B, IFRD2, HYAL3, FUS2/NAT6, HYAL1, HYAL2, FUS1/TUSC2, RASSF1/123F2, BLU/ZMYND10, NPRL2/TUSC4, 101F6/TSP10/CYB561D2, PL6/TMEM115, and CACNA2D2), and 3 open reading frames encoding hypothetical proteins (LOC100129060, LOC100287609, and C3orf45/FLJ38608).5 Most of the 19 genes demonstrate varying degrees of tumor suppressor activity (related to the control of processes such as cell differentiation, proliferation, signal transduction, and apoptosis), and it has been suggested that all function together as a large, integrated, biologically functionally diverse tumor suppressor unit.3

RBM5 (previously referred to as g15, LUCA-15, and H37) is an RNA-binding protein that has the ability to modulate apoptosis.6–13 As shown in Figure 1A, RBM5-mediated apoptosis is associated with up-regulation of the proapoptotic protein BAX, down-regulation of the antiapoptotic proteins BCL-2 and BCL-XL, increased release of mitochondrial cytochrome c into the cytosol and increased activation of caspases 9 and 3.9,11–13 RBM5 modulates apoptosis by regulating the alternative splicing of apoptosis-associated premRNAs, such as CASP2 and FAS/CD95.14,15 Although previously dismissed as unlikely to be a tumor suppressor gene, based on its lack of mutations and continued expression in most lung cancers, it has since been established that RBM5 is not alone in this characteristic, because mutations seem to be rare in the 19 genes mapping to the common deletion region.1,3,16 The fact that RBM5 was recently shown to retard tumor growth when overexpressed, albeit from a strong viral promoter, in either A9 mouse fibrosarcoma cells or A549 lung adenocarcinoma cells injected intraperitoneally into immunocompromised mice, suggests that it possesses at least some tumor suppressor activity.13,17,18 In addition, multiple protein isoforms of RBM5 exist, each possessing apoptosis modulatory activity, a function not inconsistent with tumor suppressor activity.19 (Notably, many of the other 19 genes mapping to the common deletion region, including FUS1,20 RASSF1A,21 SEMA3B,22 SEMA3F,16 HYAL1,16 and CACNA2D2,23 have the ability to modulate apoptosis.) Finally, RBM5 is 1 of 9 down-regulated genes in the 17-gene metastatic signature for solid tumors (including lung) in humans and mice, suggesting that down-regulation of the protein encoded by RBM5 is important for tumor establishment and/or progression and in a wide range of cancers.24,25

Figure 1
Figure 1
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Two smaller deletion regions at 3p21.3 have been described after analyses of homozygous deletions common to different cancer types. One deletion region, defined by Senchenko et al.,26 was common to lung cancer cell lines, renal cell carcinoma, and breast cancer biopsy samples; it included the 17 genes listed earlier but excluded RBM5 and RBM6. The second deletion region, defined by Minna et al.,27 was common to SCLC and breast cancer cell lines; it included only 9 of the 19 genes defined by the 3 SCLC homozygous deletions mentioned earlier and excluded RBM6, FUS2, and all the genes between, including RBM5. Although these data suggest that a subset of the deleted genes is associated with SCLCs and might suffice for disease progression, the contribution to malignant transformation of each of the 19 genes within the larger deletion region that is common to the 3 lung cancer cell lines, either individually or in tandem with other genes, cannot be ruled out.

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RBM5 Expression

Because loss of heterozygosity at 3p21.3 is more frequent than loss of homozygosity, it means that expression of genes mapping to this tumor suppressor region is theoretically detectable, unless expression of the remaining allele is down-regulated by a process such as promoter hypermethylation. As expected, RBM5 expression is detectable in most lung cancer cell lines and primary tissues (Table 1). The question to be answered is how is the proapoptotic and potential tumor suppressive activity of the remaining RBM5 allele silenced in lung cancers?

Table 1
Table 1
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Inactivation of potential tumor suppressor activity can be attributed to haploinsufficiency, but is more commonly a result of gene mutation or transcriptional inactivation through promoter hypermethylation (such as is the case for the 3p21.3 genes RASSF1A, BLU, SEMA3F, and SEMA3B in lung cancers).3,28 As is the case for the FUS1 tumor suppressor gene that maps to 3p21.3, only a few RBM5 mutations have been observed in lung cancer tumors, and RBM5 promoter hypermethylation does not seem to account for any observed reduced or absent expression.1,28–30 This suggests that, like FUS1, RBM5 expression and function in lung cancers are regulated at another level.

Before we conclude that promoter hypermethylation is not responsible for reduced RBM5 expression levels in lung cancer, however, it should be noted that there is evidence that promoter hypermethylation is related to both histologic subtype and smoking exposure.31 Promoter hypermethylation of specific genes was noted with a higher frequency in lung adenocarcinomas than squamous cell lung cancers.31 Within the adenocarcinoma subtype, promoter hypermethylation was more frequent in ever smokers than never smokers, and within the ever smoker subgroup, promoter hypermethylation was more frequent in current smokers than in former smokers.31 In the RBM5 promoter hypermethylation study, neither histologic subtype nor smoking history was considered.28 With reference to an earlier study by Oh et al.,17 however, it would appear that the samples used in the promoter hypermethylation study consisted of six squamous tumor samples, four adenocarcinomas, and one large cell carcinoma. The six tumor samples with the most significantly reduced RBM5 mRNA levels were of the squamous type (more often than not, associated with 3p21.3 loss and smoking, and not as frequently associated with promoter hypermethylation),1,31,32 whereas three of the nine with the less significantly reduced RBM5 mRNA levels were adenocarcinomas (associated with both smokers and never smokers, but only 50% of the time associated with 3p21.3 loss,1 and more frequently associated with promoter hypermethylation31). The one tumor sample with no change in RBM5 mRNA expression compared with its nontumor counterpart was an adenocarcinoma, whereas the one tumor sample that had more RBM5 mRNA than its nontumor counterpart was a large cell carcinoma. These results would certainly suggest that RBM5 gene expression is related to histologic subtype in NSCLC and may be related to smoking history. The results also suggest that promoter hypermethylation cannot be ruled out as an RBM5 inhibitory mechanism, because the absence of promoter hypermethylation recorded by Oh et al. may have been related to 3p21.3 homozygous deletions in at least three of the squamous tumor samples (generally smoking associated) and a nonsmoking-related tumorigenic mechanism in the adenocarcinomas. We would argue that smoking history should be investigated before definitively ruling out promoter hypermethylation as a mechanism of RBM5 expression level reduction in lung cancer.

Other than these 11 matched lung tissue samples, the only reports concerning RBM5 mRNA expression levels in lung cancer involve cell lines, most of which seem to express RBM5 at levels equivalent to levels observed in nontumor cells, a phenomenon that might be related to long-term culture-associated alterations in gene expression.1,13,18,26,29 A single study, also by Oh et al.,17 examined the levels of RBM5 protein: in a range of NSCLC primary tumor samples, the levels varied but were generally decreased compared with nontumor samples. Sixty-five percent and 82% of the squamous cell carcinoma and adenocarcinoma samples, respectively, showed a strong reduction in protein expression.

Notably, all mutation and expression studies relating to RBM5, including Northern blotting, RT-PCR, and immunohistochemistry (refer to Table 1 for references), focused on one isoform. RBM5 premRNA is alternatively spliced to produce at least three protein coding variants, some with opposing apoptotic functions.6,7,10 In addition, at least one antisense noncoding mRNA is transcribed within the RBM5 gene locus.33 Finally, differential expression of transcription-induced chimeras of RBM6 and RBM5 has been observed in tumor tissue compared with nontumor tissue and among tumor types.34 Regulation of RBM5 gene expression is, therefore, a complex process that should also be examined in relation to histologic subtype and smoking history to more clearly define the purported tumor suppressor role of RBM5.

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Smoking Status, TGF-α Signaling, and RBM5

Recent studies suggest that although all NSCLCs seem to involve alterations in the transforming growth factor (TGF)-α pathway (Figure 1B), the alterations are likely to be different depending on smoking exposure history.35 For instance, epidermal growth factor receptor (EGFR) activating mutations are more often associated with never smoker-related lung cancers, whereas KRAS activating mutations are more often associated with smoking-related NSCLC. Interestingly, RBM5 is down-regulated by the constitutively activated RAS mutant protein, RAS(G12V), in rat embryonic fibroblast cells.36 In light of these observations, it would perhaps be prudent to examine the relationship between activating EGFR mutations or activating KRAS mutations and RBM5 gene expression or function in lung cancer. In addition, one of a number of potential EGFR binding partners, the proto-oncoprotein HER2/ErbB2, seems to be overactive in a small percentage of both SCLC and never smoker-related NSCLC.37,38 These activating mutations in EGFR, KRAS, and HER2 are mutually exclusive events.39,40 Interestingly, HER2 overexpression has been shown to affect the alternative splicing of RBM5.41 In light of these recent advances regarding lung cancer signaling, we would like to suggest that future studies relating to RBM5 expression and potential tumor suppressor activity take into consideration not only the histologic subtype but also the smoking history and mutation status of genes within the TGF-α signaling pathway to get a more accurate assessment of the role played by RBM5 in lung cancer initiation and/or progression. In light of recent trends toward tailoring therapies to tumor subtype and/or genotype, this new direction for putative tumor suppressor gene functional analysis seems warranted.

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Keywords:

RBM5; LUCA-15; Apoptosis; Tumor suppressor; Gene expression; Lung cancer

© 2010International Association for the Study of Lung Cancer

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