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. 2022 May 30:12:862250.
doi: 10.3389/fonc.2022.862250. eCollection 2022.

SFPQ Promotes Lung Cancer Malignancy via Regulation of CD44 v6 Expression

Libang Yang  1 , Jianbo Yang  2   3 , Blake Jacobson  4 , Adam Gilbertsen  1 , Karen Smith  1 , LeeAnn Higgins  5 , Candace Guerrero  5 , Hong Xia  1 , Craig A Henke  1 , Jizhen Lin  3   6
Affiliations

SFPQ Promotes Lung Cancer Malignancy via Regulation of CD44 v6 Expression

Libang Yang et al. Front Oncol. .

Abstract

Mesenchymal stem cells (MSCs) contribute to tumor pathogenesis and elicit antitumor immune responses in tumor microenvironments. Nuclear proteins might be the main players in these processes. In the current study, combining spatial proteomics with ingenuity pathway analysis (IPA) in lung non-small cell (NSC) cancer MSCs, we identify a key nuclear protein regulator, SFPQ (Splicing Factor Proline and Glutamine Rich), which is overexpressed in lung cancer MSCs and functions to promote MSCs proliferation, chemical resistance, and invasion. Mechanistically, the knockdown of SFPQ reduces CD44v6 expression to inhibit lung cancer MSCs stemness, proliferation in vitro, and metastasis in vivo. The data indicates that SFPQ may be a potential therapeutic target for limiting growth, chemotherapy resistance, and metastasis of lung cancer.

Keywords: CD44v6; SFPQ; ingenuity pathway analysis; lung non-small cell (NSC) cancer; mesenchymal stem cells (MSCs); nuclear fraction; quantitative proteomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Proteomics and Ingenuity pathway analysis with lung NSC cancer and control MSC nuclear Profile. (A) Colony formation, cell proliferation and invasion assay were conducted with the normal and lung cancer MSCs. Lung NSC cancer MSCs have higher capability of proliferation(Left panel), colony forming (middle) and invasion (Right panel) than that of normal MSC cells. 4 normal lung cell lines (C210, C205, C215, C249) and 4 lung NSC cancer cell lines (Can661, Can522, Can838, A549) were used in these experiments. All data are shown as mean ± S.E. (n=3 independent experiments). (B–D) Proteins identified from control and lung NSC cancer MSC nuclear fraction with relative quantification in Proteomics analysis were applied to IPA to generate the biological networks from Lung cancer MSC and control MSCs dataset. (B) Top cell functions associated with the differentially expressed genes. (C) Top upstream regulators associated with different proteins. (D) Top canonical pathways associated with different proteins. Cell functions, upstream regulators or pathways identified are represented on the y-axis. The x-axis corresponds to the –log of the P-value (Fisher’s exact test) and the orange points on each pathway bar represent the ratio of the number of proteins in a given pathway that meet the cutoff criteria, divided by the total number of proteins that map to that pathway.
Figure 2
Figure 2
The expression level of SFPQ in lung NSC cancer MSC is higher than that in IPF and control MSC. (A) Predicted interactive proteins of SFPQ and their functional interactions are shown by IPA pathway analysis. Primary cell lines were used to measure SFPQ expression level in MSCs. The MSCs were sorted and verified from 4 normal lung cell lines (Con210, Con205, Con215, Con249) and 4 lung NSC cancer cell lines, (Can661, Can522, Can838 and A549) as described in method. (B, C) SFPQ expression was analyzed with (B) RT-PCR in mRNA and (C) western blot analysis in protein levels. Densitometry values were shown in the right graph. All data are shown as mean ± S.E. (n=3 independent experiments).
Figure 3
Figure 3
SFPQ is essential for cell stemness, proliferation, and invasion of lung NSC cancer MSCs. Lung NSC cancer MSCs isolated from A549 and Can661 cell lines were transduced with scramble or SFPQ shRNA and 48 hours later the cells were used for following analysis. (A) PARP1 mRNA level was quantitatively analyzed with RT-PCR in control and lung NSC cancer MSCs (far left: cell Con210, Con205, Con249; NSC cancer cell lines, Can661, Can838 and A549). (B) DNA damage marker H2AX was reduced in PARP1 knockdown lung cancer MSCs. γH2AX and PARP1 mRNA level in RT-PCR (left) and protein levels were analyzed with western blot analysis (middle). Densitometry analysis of WB were shown in the right graph. (C) Colony Formation Assay of lung cancer MSCs. Colony number was accounted microscopically from 6 random fields/well. Colony number was reduced in lung cancer MSCs transduced with SFPQ shRNA. (D) Sox2 expression in lung NSC cancer MSCs was quantified with RT-PCR (left panel) and western Blot analysis (middle panel). Densitometry analysis of WB are shown in the right graph. (E) Cell Proliferation assay. Lung cancer MSC proliferation was inhibited when SFPQ expression was knocked down with SFPQ shRNA. (F) Ki67 levels in lung cancer MSCs were analyzed with RT-PCR (Left) and western blot analysis (Middle). Densitometry values are shown in the right graph. (G) IC50 assay for Cisplatin. Dose responses of cisplatin were plotted as the percent of MTS staining vs. untreated cells from three replicate experiments. Lung NSC Cancer MSCs transduced with SFPQ shRNA were more sensitive to Cisplatin than the control group. (H) Cell invasion assay. Bars represent the total number of invading cells from 6 random fields/well. Invasive capacity of lung NSC cancer MSCs was decreased after SFPQ knockdown. (I) NMIIA and MMP2 expression in lung NSC cancer MSCs were quantified with RT-PCR (Left) and western blot analysis (Middle). Densitometry values are shown in the right hand graph. All data are shown as mean ± S.E. (n=3 independent experiments).
Figure 4
Figure 4
SFPQ promotes the malignant phenotype of lung NSC cancer MSCs via regulating CD44v6 expression. (A) Control and lung NSC cancer MSCs isolated from 4 normal lung cell lines (Con210, Con205, Con215, Con249) and 4 lung NSC cancer cell lines (Can661, Can522, Can838 and A549) were used to evaluate the CD44v6 levels, Which were quantified with RT-PCR (left, control vs IPF: CD44 p<0.01; CD44v6 p<0.05) and western blot analysis (middle). Densitometry analysis of WB was shown in the right graph. (B) localizations of SFPQ and CD44v6 were analyzed by the confocal microscopy with anti-SFPQ (Abcam, USA) and anti-CD44v6 (Abcam, USA) in lung NSC cancer MSCs. CD44v6 is located in both cytoplasm and nucleus. Scale Bar=20µm. (C–E) Lung NSC cancer MSCs isolated from A549 and Can 661 cell lines were transduced with scramble or SFPQ shRNA or CD44v6 shRNA Lenti virus for 48 hours, and the cells were used for following analysis: (C) Colony Assay (left), Cell proliferation assay (middle) and Cell invasion assay (right). Colony number, cell proliferation rate and invaded cells in lung NSC cancer MSCs transduced with SFPQ shRNA or CD44v6 shRNA were reduced when compared with the control group. (D) SFPQ, CD44, and CD44v6 levels were analyzed by RT-PCR (left) and western blot analysis (middle) on the same cell groups. Densitometry values are shown in the right hand graph. (E) Expression levels of Sox2, Ki67 and MMP2 were analyzed with RT-PCR (left) and western blot analysis (middle) on the same cell groups. Densitometry analysis was shown in the graph on the right. (F) mRNA level of PARP1 was analyzed with RT-PCR (left), and western blot analysis (middle) of PARP1 protein and phosphorylated γH2AX were conducted on the same cell groups. Densitometry analysis was shown in the right graph. All data are shown as mean ± S.E. (n=3 independent experiments).
Figure 5
Figure 5
SFPQ is essential for lung NSC cancer MSCs distant metastasis in vivo. NSG mice were used for cancer cell metastasis experiment. (A–N). Serial 4 µm sections of the tissues from mice receiving A549 MSCs transduced with scrambled-shRNA (A–D, I), scale bar: 200 µm; (J, K) scale bar 50 µm) or SFPQ-shRNA (E–H, L), scale bar: 200 µm; (M, N) scale bar 50 µm). Representative H&E and Trichrome staining to assess fibrosis and cancer cell nests. IHC with anti-SFPQ antibody (J, M) and anti-CD44v6 antibody (K, N) was used to assess the distribution of cells expressing SFPQ and CD44v6 in the lung tissues of mice receiving A549 MSCs transduced with scrambled shRNA or SFPQ shRNA.. (O). The tumor masses in the H&E staining from mouse lung, liver and spleen tissues were counted and summarized in figure O. Compared with mice received SFPQ-knocked down lung NSC cancer MSCs, the mice in the control group had more cancerous masses (Student T test, N=5).
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