Down-regulated Expression of SLC34A2 Was Involved in Malignant Transformation of AT-II cells in Clinic Samples
Although it had been reported that the expression of SLC34A2 in NSCLCs was significantly lower than that in their adjacent tissues[24–26], yet there had no direct report about the relative expression of SLC34A2 between NSCLCs and AT-II cells. Therefore, we first compared the expression of SLC34A2 in NSCLCs and AT-II cells in clinic samples. 57 poorly or moderately differentiated NSCLCs tissue samples were collected. And the downregulated expression of SLC34A2 was found in 45/57 collected poorly or moderately differentiated NSCLCs samples compared with their adjacent tissues (Fig. 1A). Namely, in most NSCLCs samples, the expression of SLC34A2 was found to be significantly downregulated compared with their adjacent tissues, which is consistent with our previous reports [25, 26]. Then 45 NSCLCs samples with the down-regulated expression of SLC34A2 were chosen for further study. Classically, SP-C was identified as the hallmark of AT-II cells [12, 14]. We detected the expression of SP-C by immunohistochemistry in the 45 NSCLCs tissues with down-regulated expression of SLC34A2. Cells with positive expression of SP-C were present in 18/45 NSCLCs samples (Additional File 2), indicating these 18 NSCLCs samples might be originated from the malignant transformation of AT-II cells. Secondly, the 18 NSCLCs samples with positive expression of SP-C and their adjacent tissues were chosen for further study. Referring to the previous literature [33], the corresponding adjacent tissues of the 18 NSCLCs were stained with both Napi-IIb and SP-C by double staining technology in serial sections to detect the expression of Napi-IIb in AT-II cells. The AT-II cells were cubic and located in the periphery of the alveoli [13]. In the sections, the cells with positive expression of SP-C were mostly found in the periphery of the alveoli and cuboidal. Similarly, the cells with positive expression of Napi-IIb were also mostly found in the periphery of the alveoli and their shape was cuboidal (Fig. 1B). Not only SP-C, but also SLC34A2 was identified as the hallmark of the AT-II cells recently [14]. Thus, we concluded the cells with positive expression of both Napi-IIb and SP-C were AT-II cells. Then we found that the staining intensity of Napi-IIb in the NSCLCs cells on the tumor tissue section was weaker than that in the AT-II cells on the adjacent tissue section (Fig. 1C). All these results showed the expression of SLC34A2 was downregulated in the NSCLCs cells compared with the AT-II cells. And it indicated down-regulated expression of SLC34A2 was involved in the malignant transformation of AT-II cells.
The Expressions of CD44 and CD166 Were Evaluated in NSCLCs Samples and Spheres from NSCLCs Cell Lines and NSCLCs Tissue
The primary AT-II cells were difficult to culture and maintain in a well-differentiated state [12], so it was difficult to explore how AT-II cells malignantly transformed into NSCLCs directly. According to the cancer stem cell hypothesis stated that the genetic alterations might make stem cells to malignantly transform into cancer stem cells, then cancer stem cells were differentiated into cancer cells [5]. Therefore, we supposed AT-II cells might malignantly transform into lung cancer stem cells and then differentiate into NSCLCs cells. And we intended to explore the role of SLC34A2 in the malignant transformation of AT-II cells through the lung cancer stem cells which were with AT-II cells’ characteristic.
The method for sorting lung cancer stem cells by single surface marker, such as CD133, CD326, CD44 and CD166 was controversial [28]. Researchers pointed out that it would be better to combine more than one marker to isolate CSCs from NSCLCs [29]. Evidence showed the expression of CD44 or CD166 was related to stem cell characteristics of AT-II cells [30, 31]. Thus, we speculated the lung cancer stem cells with the surface markers of CD44 and CD166 might be related to the AT-II cells. In an attempt to preliminarily identify the markers of lung cancer stem cells, 18 primary NSCLC tissue samples with the down-regulated expression of SLC34A2 compared with their adjacent tissues and positive expression of SP-C were chosen to study. They were double-stained with the surface marker CD44 and CD166 by IHC in the serial section. Some cells were detected to show both positive expressions of CD44 and CD166 in 18 out of 18 NSCLC samples (include 12 lung adenocarcinoma and 6 lung squamous cell carcinoma) (Additional File 3, Fig. 2A). As a result, it indicated that the expression of CD44+CD166+ was related to AT-II cells in NSCLCs specimens.
Furthermore, 3 NSCLC cell lines (A549, H460, and H1299) and 1 NSCLC tissue were cultured in the serum-free medium to enrich CSCs. After 2 weeks, some cells in 3 NSCLC cell lines and 1 NSCLC tissue could form spheres (Fig. 2B). And these spheres were found to possess significantly higher colony formation efficiency compared with their corresponding NSCLC cell lines and differentiated cells respectively (Fig. 2C and 2D). Then the injection of as low as 105 sphere cells (except H1299 spheres) was uncovered to consistently produce tumor xenografts in 3–5 weeks (Fig. 2E and Additional File 4). However, 106 NSCLCs cells (A549 and H460) and the differentiated cells from NSCLC tissue spheres could not generate tumor xenografts in 5 weeks (Additional File 4). All demonstrated that the spheres from NSCLC cell lines (A549 and H460) and NSCLC tissue had a higher capability of proliferation in vitro and tumorigenicity in vivo. And it indicated that these spheres contained lung cancer stem cells. Given that H1299 spheres failed to generate tumor xenografts in about 5 weeks, which pointed out the weaker CSCs characteristics of H1299, so we only took the spheres from A549, H460 and NSCLC tissue for the following study. After that, the higher proportion of CD44+CD166+ cells was found in spheres than in their corresponding NSCLC cell lines (A549 and H460) or differentiated cells by FACS (Fig. 2F). It furtherly showed that CD44+CD166+ could be the roust markers of lung cancer stem cells in spheres from NSCLCs cell lines and NSCLCs tissue.
CD44 + CD166 + Spheres from NSCLC Tissue and NSCLC Cell Lines Were Identified as Lung Cancer Stem Cells with AT-II cells’ Characteristic
To identify whether CD44+CD166+ could be a marker for lung cancer stem cells with AT-II cells’ characteristic, firstly the fractions of CD44+CD166+, CD44+CD166−, CD144−CD166+, and CD44−CD166− were respectively sorted from the spheres by FACS. Subsequently, the strongest ability of sphere formation in vitro and tumor formation in vivo was detected in CD44+CD166+ cells than CD44+CD166− and CD144−CD166+ cells (Fig. 3A,3B and Additional File 5). Moreover, the highest potential of proliferation was found in transplants of CD44+CD166+ cells by Ki-67 assay (Fig. 3C). And the mice xenografts derived by CD44+CD166+ spheres were found to show positive expression of CD44 and CD166 (Fig. 3F). Besides, CD44+CD166+ spheres had upregulated expression of the genes involved in stemness, including β-catenin, Sox2, Bmi-1, Oct-4, Nanog and CXCR4, compared with their corresponding NSCLC cell lines (A549 and H460) (Fig. 3E). And CD44+CD166+ spheres also had increased expression of the genes related to drug resistance, such as ABCG1 and Notch3, as well as the enhanced viability against cisplatin and the higher value of IC50 (Fig. 3E and Additional File 6).
After being cultured in serum-medium, CD44+CD166+ cells could differentiate into the cells which had similar morphological features with their original NSCLCs cells (Fig. 3D). At the same time, the expression of the genes involved in stemness, including β-catenin, Sox2, Bmi-1, Nanog and CXCR4, was notably reduced after CD44+CD166+ spheres were differentiated (Fig. 3E). Similarly, the expression levels of the genes related to drug resistance, e.g. ABCG1 and Notch3, as well as the viability against cisplatin and value of IC50, were also notably declined in differentiated CD44+CD166+ spheres (Fig. 3E and Additional File 6). Thus, we demonstrated a poorly differentiated state of CD44+CD166+ spheres.
The common method used for identifying the AT-II cells was to detect the expression of the surfactant protein C(SP-C) at present [14]. To explore whether CD44+CD166+ spheres were originated from AT-II cells, we detect the expression of SP-C in CD44+CD166+ spheres. It was consistent with our expectations that CD44+CD166+ spheres showed abundant expression of SP-C (Fig. 3G,3H). Also the mice xenografts derived by CD44+CD166+ spheres showed abundant expression of SP-C (Fig. 3I). These showed that CD44+CD166+ spheres were with AT-II cells’ characteristic, and indicated that CD44+CD166+ spheres might be originated from malignantly transformed AT-II cells.
Collectively, CD44+CD166+ spheres were identified as CD44+CD166+ lung cancer stem cells with the AT-II cells’ characteristic. In the following study, we named CD44+CD166+ lung cancer stem cells as LCSCs (Lung Cancer Stem Cells) for short.
SLC34A2 was Discovered to Maintain Stemness of CD44+CD166+ Lung Cancer Stem Cells in vitro and in vivo
Next, we tested the role of SLC34A2 played in LCSCs. The higher mRNA and protein expression levels of SLC34A2 were found in LCSCs than that in their corresponding NSCLC cell lines (Fig. 4A). Intriguingly, the differentiated cells that were derived from LCSCs had the lower expression of SLC34A2, compared with their LCSCs counterparts (Fig. 4A). The results above suggested that SLC34A2 might play a crucial role in maintaining stemness in LCSCs.
To verify this hypothesis, a lentiviral-based approach was used to knockdown SLC34A2 in LCSCs with si-RNAs, and the knockdown efficiency was confirmed by qRT-PCR and western blotting (Fig. 4B). Following, the weaker abilities of sphere formation, colony formation in vitro were detected in Si-SLC34A2-LCSCs than Si-NC-LCSCs and LCSCs (Fig. 4C and 4D). Importantly, when implanted into the flank of nude mice, the tumorigenicity of si-SLC34A2-LCSCs was significantly lower than NC-LCSCs and LCSCs in vivo (Figs. 4E and Additional File 7). Moreover, compared with tumor xenografts of si-NC-LCSCs and LCSCs, tumor xenografts of si-SLC34A2-LCSCs also had the weaker ability of proliferation by Ki-67 staining (Figs. 4F). Then we tested the expression of genes involved in stemness, and found the mRNA expression level of the genes related to stemness - Sox2 was declined after SLC34A2 knockdown in A549-LCSCs and H460-LCSCs (Fig. 4G). Also the mRNA expression level of Oct-4, Bmi-1, CXCR4, Nanog and β-catenin was decreased after SLC34A2 knockdown in A549-LCSCs (Fig. 4G).
Collectively, our results demonstrated that SLC34A2 was necessary to maintain the stemness of LCSCs.
SLC34A2 was Discovered to Maintain Stemness of CD44+CD166+ Lung Cancer Stem Cells by PI3K/AKT/STAT3/Sox2 Axis
Following, we intended to search the molecular mechanism under SLC34A2 maintaining stemness of LCSCs. As the mRNA expression level of Sox2 was reduced after SLC34A2 knockdown both in A549-LCSCs and H460-LCSCs, therefore, we suggested that Sox2 might play an important role in the process that SLC34A2 maintaining stemness of LCSCs. Then the declined protein expression level of Sox2 was confirmed in si-SLC34A2-LCSCs than Si-NC-LCSCs and LCSCs (Fig. 4H). Moreover, the NaPi-IIb protein, encoded by SLC34A2, was located in the cell membrane [20], thus SLC34A2 might regulate the transcription of Sox2 in nuclear by its downstream factors. Later, the reduced expression of key proteins in PI3K/AKT (PI3K and p-AKT) and STAT3 pathway(p-STAT3) was discovered in Si-SLC34A2-LCSCs than Si-NC-LCSCs and LCSCs (Fig. 5A). Additionally, significantly lower expression of key proteins (p-NF-KB and p-STAT3) in STAT3 pathway was also found in LCSCs after inhibition of PI3K/AKT pathway (LY294002) or AKT(MK2206), while inhibition of STAT3 pathway (WP1066) did not affect the expression of the key proteins (PI3K and p-AKT) in PI3K/AKT pathway in LCSCs (Fig. 5B-5E and Additional File 8). These results showed that the PI3K/AKT/STAT3 axis was located downstream of SLC34A2 in LCSCs.
After that, we aimed to find whether SLC34A2 regulated the transcription of Sox2 by the PI3K/AKT/STAT3 axis. Similar to the effect of SLC34A2 knockdown, reduced mRNA and protein expression levels of Sox2 were detected in LCSCs after inhibition of the PI3K/AKT/STAT3 axis (Fig. 5F). While significantly increased mRNA and protein expression levels of Sox2 were found in Si-SLC34A2-LCSCs after activation of the PI3K/AKT/STAT3 axis (Fig. 5G). The above showed that SLC34A2 could regulate the transcription level of Sox2 by the PI3K/AKT/STAT3 axis. Furthermore, binding sites of STAT3 to the promoter region of Sox2 were predicted via the bioinformatics prediction website (Genecards, JASPAR and Patch), and one site was confirmed by CHIP-PCR according to the reference [32] (Fig. 5H and 5I). Then, the CHIP-qPCR result uncovered the binding probability of STAT3 to the promoter region of Sox2 in LCSCs was decreased after SLC34A2 was interfered (Fig. 5J). Collectively, the above showed that SLC34A2 prompted STAT3 to bind to the promoter region of Sox2 via PI3K/AKT pathway in LCSCs.
Finally, we confirmed whether SLC34A2 maintained stemness of LCSCs via the PI3K/AKT/STAT3/Sox2 axis. Similar to the effect of SLC34A2 knockdown, the reduced stemness was found in LCSCs after inhibition of the PI3K/AKT/STAT3/Sox2 axis, such as the declined abilities of sphere formation, colony formation, growth, drug-resistance, migration, and invasion, as well as improved expression of differentiation markers CK8 and CK18 (Fig. 6A-6G and Additional File 9). While these declined abilities in si-SLC34A2-LCSCs, such as sphere formation, colony formation, growth, migration and invasion, as well as declined expression of differentiation markers CK8 and CK18, could be rescued by activation of the PI3K/AKT/STAT3/Sox2 axis (Fig. 6A-C, E-G). The ability of drug-resistance was also discovered to be largely strengthened in A549-si-SLC34A2-LCSCs after the PI3K/AKT/STAT3/Sox2 axis was activated (Fig. 6D). These results showed that SLC34A2 maintained stemness of LCSCs via the PI3K/AKT/STAT3/Sox2 axis in vitro.
The Connection between SLC34A2 Maintaining Stemness of Lung Cancer Stem Cells and PI3K/AKT/STAT3/Sox2 axis Was Validated in vivo and in clinic samples
At last, we validated the connection between SLC34A2 maintaining stemness of lung cancer stem cells and PI3K/AKT/STAT3/Sox2 axis in vivo and in clinic samples. In vivo, the weaker positive expression of key proteins in the PI3K/AKT/STAT3/Sox2 axis (PI3K, p-AKT, p-STAT3 and Sox2) was found in xenografts derived from si-SLC34A2-LCSCs than si-NC-LCSCs (Fig. 6H). Combined with our in vivo finding that Si-SLC34A2-LCSCs had reduced ability of tumorigenicity than Si-NC-LCSCs, these results indicated the connection between SLC34A2 maintaining stemness of lung cancer stem cells and the activation of the PI3K/AKT/STAT3/Sox2 axis in vivo.
As we mentioned in the first part, we found the downregulated expression of SLC34A2 in 45/57(most) poorly or moderately differentiated NSCLCs samples compared with their adjacent tissues (Fig. 1A). What's interesting was that we found the expression of SLC34A2 in LCSCs, which were enriched and originated from NSCLCs cells, was significantly higher than that in NSCLC cells in vitro (Fig. 4A). These results suggested that although the expression of SLC34A2 was significantly downregulated in NSCLC cells compared with normal cells, the lung cancer stem cells (a subset of NSCLCs) had relatively higher expression of SLC34A2 compared with other NSCLCs cells. To furtherly detect the connection between SLC34A2 maintaining stemness of lung cancer stem cells and PI3K/AKT/STAT3/Sox2 axis in clinic samples, the 45 NSCLCs samples, which had the down-regulated expression of SLC34A2 compared with their adjacent tissues, were chosen for further study. And later they were divided into two groups according to their IHC score of Napi-IIb: the group with relatively higher expression of SLC34A2(the SLC34A2High group) (IHC score of Napi-IIb ≥ 6) and the group with relatively lower expression of SLC34A2(the SLC34A2Low group) (IHC score of Napi-IIb < 6). Subsequently, data analysis displayed that the proportion of NSCLCs samples with high T stage had no significant difference between the two groups (Fig. 6I). It indicated that the downregulated expression of SLC34A2 might be associated with the early origin of NSCLCs. Besides, compared with the SLC34A2Low group, the SLC34A2High group had a higher proportion in NSCLCs samples with lymphatic metastasis and high expression of p-AKT (Fig. 6J and 6K). However, the proportion of NSCLCs samples with high expression of PI3K, p-STAT3 and Sox2 had no difference between the two groups (Fig. 6J and 6K). Cancer stem cells had a stronger ability of invasion and metastasis than cancer cells [4]. Therefore, these results preliminary suggested the connection between SLC34A2 maintaining stemness of lung cancer stem cells and the activation of AKT in the collected NSCLCs samples.
Besides, the connection between SLC34A2 maintaining stemness of lung cancer stem cells and the activation of AKT and STAT3 was also preliminarily found in the datasets of the TCGA database (Additional File 10).