The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer

Abstract

Background: SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown.

Results: Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway.

Conclusions: For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.

Fig. 1

SLC34A2 expression was down-regulated in NSCLC cell lines and SLC34A2 was re-expressed in SLC34A2-transfected cells. a mRNA levels of SLC34A2 in six NSCLC cell lines were determined by RT-PCR. HBE cells served as control. b mRNA levels of SLC34A2 in six NSCLC cell lines were detected by RT-PCR after transfected with pcDNA3.1-SLC34A2 (P-S) or pcDNA3.1 (P) for 48 h, untransfected cells was named as Control. c The concentration of phosphorous in the supernatant of medium was measured by using a phosphorous measurement kit after 48 h transient transfection

Fig. 2

SLC34A2 inhibited cell growth but had no effect on apoptosis. a Cell viability assay. A549, H1299, H460, H358, 95D and SK-MES-1 cells were transfected with pcDNA3.1-SLC34A2 or pcDNA3.1 respectively. After 48 h treatments, living cells were detected by MTT assay. b After transient transfection for 48 h, cell apoptosis ratio was determined with Annexin-V and PI staining Kit by Fluorescence-activated cell sorting (FACS)

Fig. 3

SLC34A2 inhibited migratory and invasive potential after transient transfection for 48 h. a Millicell chamber assay showed reduced cell migratory ability in SLC34A2-transfected A549, H1299, 95D and SK-MES-1 cells (P-S) compared with untransfected (Control) and vector-transfected cells (P). b Cell migration ratio of these five NSCLC cell lines was assessed by Matrigel invasion assay. c Matrigel invasion assay showed depressed cell invasive ability in SLC34A2-transfected A549, H1299, 95D and SK-MES-1 cells compared with untransfected and vector-transfected cells. d Cell invasion ratio of these five NSCLC cell lines was assessed by Millicell chamber assay

Fig. 4

SLC34A2 suppressed tumor growth and lung metastasis of NSCLC in vivo. a The expressions of SLC34A2 in A549-P-S, A549-P and A549 cells were confirmed by Western blot. b The concentrations of phosphorous in the supernatant of medium were measured by using a phosphorous measurement kit in A549-P-S, A549-P and A549 cells. c Tumor growth curve (each group contained 8 mice). d Tumor wet weight. Mice were killed for measurement of tumor weight at 60 days after inoculation. e Lungs from mice injected with A549-P-S, A549-P and A549 cells were injected intratracheally with India ink and fixed in AAF solution. f The numbers of lung nodules were counted under a dissecting microscope

Fig. 5

Relative signal pathways were determined by Western blot. There were not significant changes in the expressions of Akt, mTOR, MER1/2 and Erk1/2 in P-S (A549 and 95D) comparing with their control counterparts respectively. SLC34A2-tranfected A549 cells exhibited reduced expression levels of PI3K, p-Akt, p-mTOR, p-MEK1/2, p-Erk1/2 and CyclinD3. No significant changes in expression levels of PI3K, p-Akt, p-mTOR and CyclinD3 were found between SLC34A2- and vector- transfected 95D and 95D cells, whereas expressions of p-MEK1/2 and p-Erk1/2 in SLC34A2-transfected 95D cells were reduced (Cells transfected with pcDNA3.1-SLC34A2 or pcDNA3.1 were named as P-S or P respectively. Untransfected cells were named as Control.)