LAPTM4B is a novel diagnostic and prognostic marker for lung adenocarcinoma and associated with mutant EGFR

Lung cancer is the leading cause cancer-related death worldwide and there are approximately 85% of lung cancer being classified as non-small cell lung cancer (NSCLC) [1]. Lung adenocarcinoma (LAC) is currently the main histological subtype of NSCLC and the average 5-year survival rate of NSCLC patients remains lower than 15% [2]. Early detection and lobectomy could significantly improve survival for early stage of lung cancer [3], but for late stage patients, lack of sufficient therapeutic methods resulted in cancer exacerbation. Currently, the widely used diagnostic markers for lung cancer are pro-gastrin-releasing peptide (ProGRP), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and cytokeratin 19 fragment (CYFRA21-1) [4]. However, these tumor markers display relatively low sensitivity and specificity, so their clinical applications are limited [5, 6]. Thus, it is crucial for identification of new targets for diagnosis and monitoring treatment.

Lysosome-associated protein transmembrane-4 beta (LAPTM4B), a novel oncogene, was first identified in human hepatocellular carcinoma (HCC). LAPTM4B is tetratransmembrane lysosomal protein that is overexpressed and associated with poor prognosis in various malignancies including breast cancer, gallbladder cancer, ovarian cancer, HCC, gastric cancer and cervical cancer [7‐15]. In addition, LAPTM4B has been reported to promote proliferation and metastasis of tumor cells, resist apoptosis, initiate autophagy and assist drug resistance [16]. Recent studies showed that LAPTM4B was elevated in NSCLC and its overexpression was an independent factor in NSCLC prognosis [17, 18]. However, the clinical significance of LAPTM4B in LAC and the role of this oncogene in malignant phenotype and cell signaling remain unclear.

In this study, we first demonstrated that LAPTM4B level was significantly elevated in LAC tissues as wells as serum samples, and indicated poor survival. Further functional assays showed that LAPTM4B promoted the oncogenic phenotypes of LAC cells in vitro via PI3K/AKT and EMT signals. In addition, LAPTM4B expression level was found to be associated with EGFR gene mutations and could be influenced by mutant EGFR in LAC. Taken together, we suggest that LAPTM4B is related to LAC progression and might be a potential biomarker and theraputic target for LAC.

NSCLC is one of the most common causes of cancer related death worldwide [25]. Patients diagnosed with NSCLC are usually at late stage, and the prognosis is very poor. It’s significant to find an effective indicator to diagnose NSCLC in clinical practice. LAPTM4B was first identified in hepatocellular carcinoma and overexpressed in multiple solid tumors. In addition, LAPTM4B played critical roles in tumorigenesis and tumor metastasis in hepatocellular carcinoma, ovarian cancer, breast cancer and cervical cancer [26‐29]. Our recent efforts demonstrated that transcription factor AP4 positively regulates LAPTM4B to promote cell growth, metastasis and chemotherapy resistance in hepatocellular cancer and breast cancer [19, 30]. Based on the previous studies, we explored the biological role and clinical significance of LAPTM4B in lung adenocarcinoma.

In the present study, we sought to characterized LAPTM4B expression in LAC specimens and its impact on oncogenic phenotype of LAC cells and signaling pathways. LAPTM4B expression level was analyzed from TCGA database and found that it was overexpressed in LAC compared to normal tissues. Then, we investigate LAPTM4B protein levels in 63 LAC tissues by IHC and our results revealed that high expression of LAPTM4B correlated with aggressive clinicopathological features (including advanced clinical stages, lymph node metastasis and EGFR mutations). Moreover, our data also identified that OS and PFS of LAC patients were significantly worse in the LAPTM4B high expression group compared to the low expression group and the result was consistent with the previous study reporting that LAPTM4B was associated with poor survival in LAC but not in all NSCLC patients [17].

LAPTM4B is a type III transmembrane protein with four putative transmembrane regions and highly expressed in LAC tissues, so we test whether serum levels of LAPTM4B could be used as a tumor marker in LAC. This is the first report investigating the value of serum LAPTM4B levels as a biomarker for the patients with LAC. Our results demonstrated that serum levels of LAPTM4B were much higher in a relatively large series of LAC samples (n = 216) than those in the healthy controls. Furthermore, serum LAPTM4B levels were positively correlated with smoking, advanced clinical stages, lymph node metastasis, ALK rearrangements and EGFR mutations. In addition, we evaluated the value of serum LAPTM4B levels in chemotherapy response of LAC patients. The results showed that serum LAPTM4B levels were significantly decreased after chemotherapy. We also compared LAPTM4B levels in different chemotherapy efficacy groups and found that serum LAPTM4B levels of chemotherapy patients with PD and SD were much higher than those with PR and CR, suggesting LAPTM4B have the potential to predict chemotherapy response of LAC patients. EGFR gene mutations are detected in 30 to 40% of patients with NSCLC in China and associated with poor prognosis [31]. Patients with EGFR mutations are highly sensitive to EGFR-TKIs [32] but approximately 10% of NSCLC patients harboring EGFR mutations exhibit primary resistance [33]. Our study demonstrated that high LAPTM4B expression was associated with EGFR-TKI resistance in LAC patients with EGFR mutations. Additional large-cohort studies are needed to confirm the finding. ROC curve analysis revealed that the sensitivity and specificity of LAPTM4B were 75.6 and 82.5%, respectively. Serum LAPTM4B level showed an early diagnostic performance comparable to CEA and CYFR 21–1 [34].

Our in vitro findings revealed that overexpression of LAPTM4B promoted, while silencing LAPTM4B inhibited proliferation, migration and invasion of LAC cells. To further clarify the mechanism of LAPTM4B involvement in cell growth and migration, the associated proteins of PI3K/AKT and EMT signals in LAC cells were investigated. Western blotting results showed that the two signals were activated in LAPTM4B overexpression cells. It is worthwhile to mention that previous report has shown that the PPRP motif contained in the N-terminus of LAPTM4B-35 could interact with the p85α regulatory subunit of PI3K which results in activation of PI3K/AKT signals. PI3K/AKT signaling pathway participated in LAPTM4B-promoting tumor progression and multidrug resistance in hepatocellular carcinoma, breast cancer and prostate cancer [19, 26, 30, 35]. The current findings along with the previous published reports suggest that LAPTM4B/AKT signaling axis may present a novel target for the treatment of LAC. Additionally, c-myc regulates cell growth, differentiation, apoptosis and motility, and is involved in the pathogenesis of many cancers [36, 37]. In the study, we also found that c-myc protein was significantly increased by LAPTM4B overexpression and may play an important role in LAC progression.

Notably, we found that high LAPTM4B expression was associated with EGFR mutations in LAC tissues and serum samples. Moreover, A549 cell with EGFR-wild type exhibited relatively lower LAPTM4B expression compared to H1299 cell line displaying high invasiveness and other cell lines (H1975, PC9 and HCC827) with EGFR mutations. These observations led us to investigate the association between LAPTM4B expression and EGFR-activating mutations. Of note, activating mutations within the EGFR tyrosine kinase domain including in-frame deletions in exon 19 and L858R point mutations in exon 21 were found to be predictors of clinical response to EGFR-TKIs [38]. Our results showed that EGFR-TKIs significantly decreased LAPTM4B expression in HCC827 (EGFR exon 19 deletion mutation) and H1975 (L858R point mutation and secondary T790 M mutation) cell lines, insinuating that LAPTM4B may be affected by EGFR kinase activity. Similarly, earlier work has demonstrated that EGFR was co-immunoprecipitated with endogenous LAPTM4B and LAPTM4B overexpression correlates with EGFR activation in gastric cancer cells [39]. Moreover, Tan X et al. reported that upon serum starvation, LAPTM4B senses EGFR inactivation at endosomes and selectively forms a complex with inactive EGFR to initiate autophagy [40]. On the other hand, LAPTM4B interacts with Nedd4 through its PY motifs at the LAPTM4B C-tail and further enhance the ubiquitination of Hrs, which could block EGF-stimulated EGFR intraluminal sorting and degradation. Finally, EGFR signaling could be prolonged and exert influence on tumor progression [41]. Here, in vitro assays revealed that LAPTM4B could enhance the effects of EGFR on cell proliferation, migration and invasion. Together with our findings, LAPTM4B may interact with EGFR and play an important role in the pro-survival functions of EGFR in cancer cells.

As we known, activation of the PI3K-AKT-mTOR axis is more robustly induced by mutant EGFR than by wild-type EGFR. Phosphorylated EGFR being the docking site of PI3K stimulates the activation of AKT [42], and then activates the downstream target molecule mTOR to induce the expression of cell cycle-related proteins and promote cells G1-S transition [43]. Then we further delineated the role of this signaling pathway in regulating LAPTM4B by mutant EGFR. Our result revealed that LY294002, the PI3K inhibitor, could suppress LAPTM4B expression in LAC cells with EGFR mutations. Moreover, previous studies confirmed that EGFR-TKIs can block the PI3K/AKT signaling pathway by inhibiting the kinase activity of mutant EGFR, which have been implicated in the inhibition of cell apoptosis and the promotion of cell growth and motility [44, 45]. However, among patients with advanced EGFR-mutated NSCLC, treatment with EGFR-TKIs (e.g., gefitinib, erlotinib, and afatinib) is associated with response rates of 56 to 74% and a median progression-free survival of 10 to 14 months. In addition, the majority of patients will have disease progression within 1 to 2 years after treatment initiation due to the acquired resistance [46‐49]. Our results showed that LAPTM4B overexpression could significantly inhibit cell apoptosis induced by gefitinib and make HCC827 cell line harboring EGFR mutations more resistant to EGFR-TKIs. Thus, it is plausible to propose that targeting LAPTM4B and PI3K/AKT pathway may help augment the effects of EGFR-TKIs treatment.

There are several potential limitations in our study. The relative small size population were enrolled in the study, and larger collaborative studies needed to validate our report. In addition, the roles of LAPTM4B on the efficacy of EGFR-TKIs treatment in LAC should be further elucidated.

In summary, our results revealed that LAPTM4B was overexpressed in LAC and high LAPTM4B expression indicated poor prognosis in patients with LAC. The expression level of LAPTM4B in the serum of patients with LAC is significantly associated with the tumor progression and treatment effects, suggesting that detection of serum LAPTM4B will facilitate the diagnosis and better predict treatment response. Further functional assay indicated LAPTM4B plays an important role in tumor proliferation and metastasis in LAC. In addition, LAPTM4B may take part in EGFR-mediated cell pro-survival and gefitinib resistance of LAC cells with EGFR mutations. These fruitful works indicate that LAPTM4B might be an important biomarker and a novel therapeutic target for LAC.