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CXCR6 expression in non-small cell lung carcinoma supports metastatic process via modulating metalloproteinases.

Mir H, Singh R, Kloecker GH, Lillard JW, Singh S - Oncotarget (2015)

Bottom Line: High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined.Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls.Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA, USA.

ABSTRACT
Lung cancer (LuCa) is the leading cause of cancer-related deaths worldwide regardless of the gender. High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined. Here, we present evidence that chemokine receptor CXCR6 and its only natural ligand, CXCL16, are significantly expressed by non-small cell lung cancer (NSCLC) and are involved in the pathobiology of LuCa. CXCR6 expression was significantly higher in two subtypes of NSCLC (adenocarcinomas-ACs and squamous cell carcinoma-SCCs) as compared to non-neoplastic tissue. Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls. Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients. Biological significance of this axis was validated using SCC and AC cell lines. Expression of CXCR6 was higher in AC cells, which also showed higher migratory and invasive potential than SCC. Differences in migratory and invasive potential between AC and SCC were due to differential expression of metalloproteinases following CXCL16 stimulation. Hence, our findings suggest clinical and biological significance of CXCR6/CXCL16 axis in LuCa, which could be used as potential prognostic marker and therapeutic target.

No MeSH data available.


Related in: MedlinePlus

Expression of MMP and TIMP in LuCa cells(A) Changes in mRNA levels of MMPs and TIMP after treatment with 100 ng/ml CXCL16 for 30 minutes. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective untreated cells or as indicated. (B) Percent increase in the levels of secreted MMP-2, 9 and TIMP-2 proteins 24 h after stimulation with 100 ng/ml CXCL16. *p ≤ 0.05 compared to respective untreated cells. Levels of MMP-2 in AC (NCI-H2126) and MMP-9 in SCC (NCI-H520) were below detection limit of the kit. (C) Basal levels of MMP and TIMP mRNA in SCC (NCI-H520) and AC (NCI-H2126) cells. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to SCC. MMP-9 mRNA could not be detected in SCC cells.
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Figure 5: Expression of MMP and TIMP in LuCa cells(A) Changes in mRNA levels of MMPs and TIMP after treatment with 100 ng/ml CXCL16 for 30 minutes. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective untreated cells or as indicated. (B) Percent increase in the levels of secreted MMP-2, 9 and TIMP-2 proteins 24 h after stimulation with 100 ng/ml CXCL16. *p ≤ 0.05 compared to respective untreated cells. Levels of MMP-2 in AC (NCI-H2126) and MMP-9 in SCC (NCI-H520) were below detection limit of the kit. (C) Basal levels of MMP and TIMP mRNA in SCC (NCI-H520) and AC (NCI-H2126) cells. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to SCC. MMP-9 mRNA could not be detected in SCC cells.

Mentions: Matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) play crucial role in dissemination and invasion of tumor cells. Studies have shown MMP-1, -2, -9, -11 and -14 are highly expressed by LuCa [27–31]. Hence, we analyzed levels of these MMPs in LuCa cell lines with respect to CXCR6-CXCL16 axis (Figure 5A–5C). MMP-2 transcripts increased in both NSCLC cell lines (AC and SCC) after CXCL16 stimulation; moreover, the increase in AC was significantly higher than in SCC. In addition to this, MMP-11 and -14 mRNAs were also elevated in SCC after CXCL16 stimulation. However, MMP-9 mRNA was undetectable in SCC while, AC showed a significant increase in MMP-9 mRNA after CXCL16 stimulation. Interestingly, MMP-1, -11, -14 mRNAs in AC did not change after CXCL16 addition. CXCL16 treatment also increased MMP-2 protein levels to ~108.6% in SCC conditioned media collected 24 h after the treatment, but it was below the detection limit in conditioned media collected from AC after CXCL16 stimulation. There was marginal increase in MMP-9 (~16.7%) levels in conditioned media from AC cells after CXCL16 stimulation but its was below the detection limit in SCC conditioned media after CXCL16 stimulation. We also analyzed expression of TIMPs in LuCa cells following CXCL16 treatment. We could not detect TIMP-1 mRNA or protein in AC and SCC cell lines used in this study. Levels of TIMP-2 mRNA remained unchanged in both cell lines (Figure 5A). Furthermore, the change in TIMP-2 protein in SCC after 24 h CXCL16 treatment was significantly higher (~32.4%) (p ≤ 0.05) (Figure 5B). In contrast, there was no significant change in TIMP-2 protein in AC cells after CXCL16 treatment.


CXCR6 expression in non-small cell lung carcinoma supports metastatic process via modulating metalloproteinases.

Mir H, Singh R, Kloecker GH, Lillard JW, Singh S - Oncotarget (2015)

Expression of MMP and TIMP in LuCa cells(A) Changes in mRNA levels of MMPs and TIMP after treatment with 100 ng/ml CXCL16 for 30 minutes. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective untreated cells or as indicated. (B) Percent increase in the levels of secreted MMP-2, 9 and TIMP-2 proteins 24 h after stimulation with 100 ng/ml CXCL16. *p ≤ 0.05 compared to respective untreated cells. Levels of MMP-2 in AC (NCI-H2126) and MMP-9 in SCC (NCI-H520) were below detection limit of the kit. (C) Basal levels of MMP and TIMP mRNA in SCC (NCI-H520) and AC (NCI-H2126) cells. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to SCC. MMP-9 mRNA could not be detected in SCC cells.
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Figure 5: Expression of MMP and TIMP in LuCa cells(A) Changes in mRNA levels of MMPs and TIMP after treatment with 100 ng/ml CXCL16 for 30 minutes. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective untreated cells or as indicated. (B) Percent increase in the levels of secreted MMP-2, 9 and TIMP-2 proteins 24 h after stimulation with 100 ng/ml CXCL16. *p ≤ 0.05 compared to respective untreated cells. Levels of MMP-2 in AC (NCI-H2126) and MMP-9 in SCC (NCI-H520) were below detection limit of the kit. (C) Basal levels of MMP and TIMP mRNA in SCC (NCI-H520) and AC (NCI-H2126) cells. Values are mean ± SEM from 3 independent experiments. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to SCC. MMP-9 mRNA could not be detected in SCC cells.
Mentions: Matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) play crucial role in dissemination and invasion of tumor cells. Studies have shown MMP-1, -2, -9, -11 and -14 are highly expressed by LuCa [27–31]. Hence, we analyzed levels of these MMPs in LuCa cell lines with respect to CXCR6-CXCL16 axis (Figure 5A–5C). MMP-2 transcripts increased in both NSCLC cell lines (AC and SCC) after CXCL16 stimulation; moreover, the increase in AC was significantly higher than in SCC. In addition to this, MMP-11 and -14 mRNAs were also elevated in SCC after CXCL16 stimulation. However, MMP-9 mRNA was undetectable in SCC while, AC showed a significant increase in MMP-9 mRNA after CXCL16 stimulation. Interestingly, MMP-1, -11, -14 mRNAs in AC did not change after CXCL16 addition. CXCL16 treatment also increased MMP-2 protein levels to ~108.6% in SCC conditioned media collected 24 h after the treatment, but it was below the detection limit in conditioned media collected from AC after CXCL16 stimulation. There was marginal increase in MMP-9 (~16.7%) levels in conditioned media from AC cells after CXCL16 stimulation but its was below the detection limit in SCC conditioned media after CXCL16 stimulation. We also analyzed expression of TIMPs in LuCa cells following CXCL16 treatment. We could not detect TIMP-1 mRNA or protein in AC and SCC cell lines used in this study. Levels of TIMP-2 mRNA remained unchanged in both cell lines (Figure 5A). Furthermore, the change in TIMP-2 protein in SCC after 24 h CXCL16 treatment was significantly higher (~32.4%) (p ≤ 0.05) (Figure 5B). In contrast, there was no significant change in TIMP-2 protein in AC cells after CXCL16 treatment.

Bottom Line: High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined.Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls.Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, GA, USA.

ABSTRACT
Lung cancer (LuCa) is the leading cause of cancer-related deaths worldwide regardless of the gender. High mortality associated with LuCa is due to metastasis, molecular mechanisms of which are yet to be defined. Here, we present evidence that chemokine receptor CXCR6 and its only natural ligand, CXCL16, are significantly expressed by non-small cell lung cancer (NSCLC) and are involved in the pathobiology of LuCa. CXCR6 expression was significantly higher in two subtypes of NSCLC (adenocarcinomas-ACs and squamous cell carcinoma-SCCs) as compared to non-neoplastic tissue. Additionally, serum CXCL16 was significantly elevated in LuCa cases as compared to healthy controls. Similar to CXCR6 tissue expression, serum level of CXCL16 in AC patients was significantly higher than SCC patients. Biological significance of this axis was validated using SCC and AC cell lines. Expression of CXCR6 was higher in AC cells, which also showed higher migratory and invasive potential than SCC. Differences in migratory and invasive potential between AC and SCC were due to differential expression of metalloproteinases following CXCL16 stimulation. Hence, our findings suggest clinical and biological significance of CXCR6/CXCL16 axis in LuCa, which could be used as potential prognostic marker and therapeutic target.

No MeSH data available.


Related in: MedlinePlus