Introduction
Lung cancer remains the main cause of cancer-related deaths worldwide (1). Cases of non-small cell lung cancer (NSCLC) in young patients (≤50 years old) represent a small percentage of the total cases, and indeed this disease typically affects older individuals (>50 years old), and the incidence rate among elderly patients is increasing (2). Previous studies have compared young and aged NSCLC patients, using the range of 40 to 50 years to define the young group (2–8); however, to date, it is not clear if lung cancer, particularly adenocarcinoma, in young patients may have distinct clinicopathological features. In the present paper, liver kinase B1 (LKB1) and its downstream signalling pathways were investigated as a therapeutic target in lung adenocarcinoma, a subtype of NSCLC (9), and compared between different age groups. LKB1, also known as serine/threonine kinase 11, is a tumor suppressor gene involved in cellular responses including growth, polarity and metabolism (10). LKB1 is a master kinase, controlling 14 substrates involved in the translation of several cell growth regulators (11). LKB1 was been initially identified as the tumor suppressor responsible for Peutz-Jeghers syndrome, an inherited cancer predisposition (12). Several sporadic tumors exhibited LKB1 promoter hypermethylation, reduced LKB1 expression and somatic LKB1 mutations, indicating a role of the loss of LKB1 in cancer development and progression, potentially with additional oncogenic factors (13). LKB1 may also be repressed as a result of post-transcriptional regulation by microRNAs (miRNA/miRs) (10), which generally serve to repress mRNA translation or promote mRNA degradation via partial complementary binding to the 3′ untranslated region (3′-UTR) of target mRNAs (14).
The role of LKB1 in NSCLC has previously been analysed; however, study is made difficult by the fact that the LKB1 pathway involves multiple substrates that act on metabolism, apoptosis and the tumor microenvironment. Carretero et al (15) reported that NSCLC cells with loss of LKB1 exhibited higher nuclear expression of catenin β-1 (CTNNB1); LKB1, in fact, appears to suppress the Wnt/CTNNB1 pathway, inhibiting the expression of downstream genes, including cyclin D1 (CCND1) and survivin. Additionally, lysyl oxidase (LOX) has been reported to be negatively regulated by LKB1 in lung cancer, and yes-associated protein 1 (YAP1) has been reported to be activated in lung adenocarcinoma as a result of a lack of LKB1 (16). In the present study, the mRNA expression of LKB1, CCND1, CTNNB1, LOX, YAP1, survivin and 15 miRNAs involved in the LKB1 pathway was investigated using NanoString technology. LKB1 loss has been reported to be more common within KRAS-mutant lung adenocarcinomas (17), and therefore, KRAS mutations were also investigated.
The results presented provide indication that LKB1 pathway genes, with the involvement of miRNA regulation, may have a role in lung adenocarcinoma progression, representing novel potential targets for lung cancer therapy.
Discussion
Lung cancer remains the main cause of cancer-related mortality worldwide, and the age at diagnosis has been decreasing in recent years (2,4). Younger patients with lung cancer appear to exhibit distinct clinicopathological features: They are more commonly non-smokers and female, and present a prevalence for adenocarcinoma and advanced disease; however, there is controversy regarding the outcome as it has been reported as improved by certain studies (4,31) and unaffected by others (31–34). Data on the management of adenocarcinoma in the elderly are insufficient (35–39), and thus, whether young lung cancer patients have specific molecular and pathologic features or different survival outcomes remain unclear. In the present study, 44 lung adenocarcinoma patients ≤50 years old were selected as the younger group and 44 cases >50 years as the older group; a predominance of females was identified in the younger group, and the acinar pattern was most prevalent, which was in accordance with previous studies (4,40–42). There were no significant differences in survival, in terms of DFI and OS, in young lung adenocarcinoma patients compared with the older age group. At present, there is no general consensus on the influence of age on survival, and this issue is open to question. Any discrepancies between reports may be due to the limited number of studies and to the specific cut-off age used to separate younger from older patients; the current study used 50 years old as the cut-off value, according to several previous reports (2,43,44).
An aim of this retrospective study was also to focus on the expression pattern of LKB1 and its downstream signalling pathways, in order to evaluate their associations with clinicopathological features and prognoses in lung adenocarcinoma, comparing younger patients with their elderly counterparts.
Although there are currently no drugs in routine clinical use that specifically target LKB1, there is a growing number of approaches that may differentially benefit patients with a dysregulated LKB1 pathway (45–49). A critical role for LKB1 has been suggested in catenin-beta1 signalling in lung cancer through its modulation of CCND1 and survivin gene expression (50). Additionally, LOX has been reported to be efficiently suppressed by LKB1, and YAP1 has been reported to be initially activated by LKB1 loss in lung ADC.
A mRNA panel was customized of the 6 abovementioned genes and 15 miRNAs involved in the LKB1 pathway, and expression profiling was performed with NanoString technology, a recently developed platform that can make direct multiplexed measurements of expression through digital readouts of the abundance of mRNA/miRNA transcripts (51). Several studies have indicated that the NanoString technique is a reliable and flexible method for the assessment of gene expression in limited FFPE tissues, and it has exhibited similar results using fresh-frozen tissue (52–54). This field appears to be of importance, since FFPE represents most of the specimens collected in routine diagnostic pathology, and it will allow for the connection of clinical follow-up data to large numbers of patients (55); therefore, the NanoString methodology may be readily adapted clinically as a highly reproducible alternative to quantitative polymerase chain reaction and sequencing methods (56).
LKB1 has different molecular targets, and thus, screening of the LKB1 signalling pathway appears a necessary step to explain its suppressive function in cancer cell biology. No differences in LKB1 levels and KRAS mutation rates were identified between young and older patients, possibly due to the biological heterogeneity of KRAS-mutant lung adenocarcinomas, to the relatively small group size, and/or to other underlying molecular differences such as epidermal growth factor mutations or anaplastic lymphoma kinase translocations (18). Notably, low LKB1 expression was apparent in the solid histological subtype, and high expression in females and early clinical stage, which suggest an important role for LKB1 in inhibiting the growth of lung cancer cells, considering that the solid subtype, male gender and advanced stages are reportedly survival disadvantages in lung adenocarcinoma (57). However, it remains unclear how LKB1 loss contributes to lung carcinogenesis, and the post-transcriptional regulation of LKB1 may play a central role. The current results also predicted that miR-93 may be able to downregulate LKB1 and CCND1, consequently leading to the loss of LKB1-dependent tumor suppression; such is in agreement with previous studies reporting that high levels of miR-93 (58) and low levels of LKB1 (59) as well as CCND1 (60) were correlated with poor survival among lung cancer patients.
The current study also attempted to elucidate the involvement of LOX. It is reported that the tumor microenvironment serves a critical role in tumorigenesis (61), and that LOX, due to its influence on the cellular microenvironment, may be a target for cancer therapy (62,63). The current data further indicated that aberrant LOX expression was involved in lung carcinogenesis and cancer progression, revealing that LOX levels in adenocarcinoma patients were significantly associated with overall stage distribution and poor prognosis regardless of age at diagnosis. Furthermore, the findings indicated that the observed positive prognostic effect of LOX was associated, at least in part, to miR-30b regulation, confirming the conclusions by Zhong et al (64) regarding a central role of this miRNA in NSCLC suppression. However, the impact of LOX remains incompletely clear; it is possible that there exists multiple forms of LOX proteins (65), and it is not known whether the signalling components downstream of LKB1, including LOX, may be involved in an LKB1-independent manner; further studies will be required to reach a conclusive point.
The finding that LKB1 and LOX may be repressed by specific miRNAs establishes a regulatory link within the LKB1 tumor suppressor pathway; miRNA-dependent post-transcriptional regulation of LKB1 may be an alternative to inactivating LKB1 mutations, which are rare among sporadic tumors (10). Additional study on LKB1 pathways and other components of the LKB1 complex may expand knowledge regarding tumor metabolism and growth potential in lung adenocarcinoma.