Recent efforts to elucidate genetic factors in lung cancer susceptibility have identified the chromosome 15q24-25.1 locus (9
). The 15q24-25.1 locus contains an iron metabolism protein gene IREB2
, a 20S proteasome structural protein gene PSMA4
, three nAChR subunit genes CHRNA5, CHRNA3
, and CHRNB4
, and a hypothetical gene LOC123688
. An ongoing question is which among the six candidates accounts for an increased risk of lung cancer. In the current study, we have fine-mapped the 15q24-25.1 locus into two subregions: hapL
for IREB2, LOC123688
, and PSMA4
; and hapN
for CHRNA5, CHRNA3
, and CHRNB4
. Our in vitro
investigation into the genes around hapL
as modulators of tumor cell proliferation implicated PSMA4
as a strong candidate. This result is physiologically relevant for two reasons. First, there is endogenous expression of PSMA4
in the two human cancer cell lines used in this study, and second, expression of PSMA4
is upregulated in lung cancer as well as in other cancer types.
At present, there is no evidence that SNP status of these genes is directly linked to the mitogenic activity of their protein products. Haplotype or SNP data neither reflect genetic changes (such as at the mRNA/protein expression level or protein function) of the candidate gene(s) in detail, nor tell whether these genetic changes could alter biological functions. Identifying causal genetic variants in the candidate gene(s) is very challenging and requires further extensive genetic and biological investigations. Any genetic change will eventually be exhibited at the mRNA/protein level or protein function to play biological function. Therefore, in vitro changing gene expression levels via overexpression/knockdowns and assaying for proliferation and apoptosis would be more efficient and informative. By these approaches, we have shown that PSMA4 affects lung cancer cell proliferation and apoptosis. This result suggests that PSMA4 is a candidate lung cancer susceptibility gene in the 15q24-25.1 locus, although the other five candidate genes in this high linkage disequilibrium region cannot be completely excluded. Our in vitro analyses were limited to human lung cancer (lung cancer cell lines and lung cancer samples). However, the role of PSMA4 in leading tumorigenesis may not be limited to lung cancer. Similar to other oncogene/tumor suppressor genes, such as P53 and PTEN, elevated expression of PSMA4 is also found in other human cancer types.
The 26S proteasome contains one 20S proteasome core and two 19S regulatory caps. The 20S proteasome core is hollow and forms an enclosed cavity where proteins are degraded. The 20S proteasome core contains two types of subunits: α subunits are structural and serve as docking domains for proteasome assembly and regulators of proper function; β subunits are predominantly catalytic (14
). PSMA4, a proteasome α type subunit 4, has been characterized as a structural subunit of the 20S proteasome core (14
). Our preliminary biological analysis revealed that PSMA4 governs proteasome activity. We showed in vitro
that knockdown of PSMA4
expression decreases proteasome activity and results in the accumulation of ubiquitinated proteins. This proteasome is responsible for the degradation of proteins involved in the activation or repression of many cellular processes, including transcription, cell-cycle progression, and apoptosis. Therefore, proteasome dysfunction stresses cells. In our case, knockdown of PSMA4
expression in cancer cells induced apoptosis. Cancer cells show increased proliferation and decreased apoptosis in vivo
via various mechanisms. Our findings that the down-regulation of PSMA4
expression induces apoptosis and that PSMA4
expression is increased in nearly half of the lung cancers studied suggest that upregulation of proteasome activity may be a novel mechanism of tumorigenesis. Indeed, inhibition or down-regulation of proteasome activity by proteasome inhibitors has already been adopted as a form of cancer treatment (20
). Therefore, our findings strongly suggest that PSMA4
plays a direct role in cancer cell proliferation.
We also systemically evaluated the nAChR subunit genes, particularly CHRNA5
, to determine if any of them has a regulatory effect on lung cancer cell growth. Recent studies have linked rs16969968, a missense SNP (D398N) in CHRNA5
, to smoking, nicotine addiction, and lung cancer (12
). We overexpressed CHRNA5-D398 in parallel with CHRNA5-N398 (rs16969968) in A549 and H1299 cells and did not observe any difference in growth (data not shown). Collectively, our current biological analysis of the nAChR subunit genes around hapN
did not reveal any growth or apoptotic differences between normal lung and lung cancer cells. Our data strongly suggest that these nAChR subunit genes are not directly involved in lung cancer, but may instead affect nicotine dependence and propensity to smoke and thus indirectly increase the likelihood of developing lung cancer.
In summary, we have shown for the first time that PSMA4 at the 15q24-25.1 locus plays a direct role in regulating lung cancer cell growth. Further understanding of the molecular mechanisms of the candidate genes may result in the development of new strategies for prevention, early detection, diagnosis, and treatment of lung cancer and other human cancers.