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PDL1 Regulation by p53 via miR-34.

Cortez MA, Ivan C, Valdecanas D, Wang X, Peltier HJ, Ye Y, Araujo L, Carbone DP, Shilo K, Giri DK, Kelnar K, Martin D, Komaki R, Gomez DR, Krishnan S, Calin GA, Bader AG, Welsh JW - J. Natl. Cancer Inst. (2015)

Bottom Line: Finally, miR-34a delivery reduced the numbers of radiation-induced macrophages (mean of F4-80 expression percentage of control group = 52.4%, SD = 1.7%; mean of F4-80 expression percentage of MRX34 = 40.1%, SD = 3.5%, P = .008, n = 4) and T-regulatory cells.We identified a novel mechanism by which tumor immune evasion is regulated by p53/miR-34/PDL1 axis.Our results suggest that delivery of miRNAs with standard therapies, such as XRT, may represent a novel therapeutic approach for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Radiation Oncology (MAC, DV, XW, YY), Experimental Therapeutics (CI, GAC), and Radiation Oncology (RK, DRG, SK, JWW), The University of Texas MD Anderson Cancer Center, Houston, TX; Mirna Therapeutics, Inc., Austin, TX (HJP, KK, DM, AGB); Ohio State University, Columbus, OH (LA, DPC, KS); Texas Veterinary Pathology Associates (Houston), Houston, TX (DKG).

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miR-34s regulation of PDL1. Endogenous levels of miR-34a, -b, and -c in non–small cell lung cancer (NSCLC) cell lines. A) PDL1 expression in A549, H460, and H1299 cells transfected with miR-34a, -b, and -c. B-J) NSCLC cell lines were treated with miR-34a, miR-34b, or miR-34c at 100nM, and 24 hours later RNA was isolated to study miR-34a, -b, and -c transfection efficiency. At 96 hours after transfection, cell lysates were collected for protein analysis. Quantification of western blots shows that forced overexpression of miR-34a, miR-34b, or miR-34c suppressed PDL1 protein expression compared with a scrambled control. K) Luciferase activity in cells cotransfected with miR-34a, -b, or -c or a scrambled control and a luciferase reporter construct encoding the luciferase gene fused either to the wild-type PDL1 3’ UTR (PDL1 wt) or a mutated PDL1 3’ UTR (PDL1 mut). All three of the miR-34s reduced luciferase activity (P < .001, P =.006, and P =.006). An unpaired t test was used to calculate the two-sided P values. *P < .05, **P < .01, ***P < .001. Error bars on the bar charts represent standard deviation. Similar results were observed in three replicates. mut = mutated; NSCLC = non–small cell lung cancer; scr = scrambled; wt = wild-type.
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Figure 3: miR-34s regulation of PDL1. Endogenous levels of miR-34a, -b, and -c in non–small cell lung cancer (NSCLC) cell lines. A) PDL1 expression in A549, H460, and H1299 cells transfected with miR-34a, -b, and -c. B-J) NSCLC cell lines were treated with miR-34a, miR-34b, or miR-34c at 100nM, and 24 hours later RNA was isolated to study miR-34a, -b, and -c transfection efficiency. At 96 hours after transfection, cell lysates were collected for protein analysis. Quantification of western blots shows that forced overexpression of miR-34a, miR-34b, or miR-34c suppressed PDL1 protein expression compared with a scrambled control. K) Luciferase activity in cells cotransfected with miR-34a, -b, or -c or a scrambled control and a luciferase reporter construct encoding the luciferase gene fused either to the wild-type PDL1 3’ UTR (PDL1 wt) or a mutated PDL1 3’ UTR (PDL1 mut). All three of the miR-34s reduced luciferase activity (P < .001, P =.006, and P =.006). An unpaired t test was used to calculate the two-sided P values. *P < .05, **P < .01, ***P < .001. Error bars on the bar charts represent standard deviation. Similar results were observed in three replicates. mut = mutated; NSCLC = non–small cell lung cancer; scr = scrambled; wt = wild-type.

Mentions: The inverse correlation of miR-34 and PDL1 expression in NSCLC cells and in tumor samples implicated miR-34 as a regulator downstream of p53 to repress PDL1. This hypothesis was further corroborated by the observation that the 3’ untranslated region (UTR) of the PDL1 mRNA carries a putative miR-34 binding site at position 932–938 (32,33). We first analyzed endogenous levels of miR-34a, -b, and -c in NSCLC cell lines with different genetic backgrounds (Figure 3A). In agreement with our findings in NSCLC patient samples (Supplementary Figure 1, available online), miR-34a expression levels were higher than miR-34b and miR-34c levels in all three NSCLC cell lines. As shown in Figure 3, B-G, enforced overexpression of miR-34a suppressed the expression of PDL1 protein compared with a scrambled control. In addition, enforced overexpression of miR-34b or miR-34c suppressed the expression of PDL1 protein compared with a scrambled control (Figure 3, H-J). Next, to determine whether miR-34 interacts directly with the putative target gene PDL1, we cotransfected H1299 cells with miR-34a, -b, or -c mimics and a reporter vector encoding the luciferase gene that is fused to the 3′ UTR of the PDL1 gene (luc-PDL1). As shown in Figure 3K, luciferase activity was reduced in cells transfected with miR-34 and the luc-PDL1 construct compared with scrambled controls. In contrast, mutation of the predicted miR-34 binding site in the 3’ UTR of PDL1 rescued the luciferase activity, thus confirming that miR-34a, miR-34b, and miR-34c interact directly with the PDL1 3′ UTR (Figure 3K) (fold-change luciferase activity, miR-34a mean = 0.50, SD = 0.2, P < .001; miR-34b mean = 0.52, SD = 0.2, P = .006; and miR-34c mean = 0.59, SD = 0.14, P = .006).


PDL1 Regulation by p53 via miR-34.

Cortez MA, Ivan C, Valdecanas D, Wang X, Peltier HJ, Ye Y, Araujo L, Carbone DP, Shilo K, Giri DK, Kelnar K, Martin D, Komaki R, Gomez DR, Krishnan S, Calin GA, Bader AG, Welsh JW - J. Natl. Cancer Inst. (2015)

miR-34s regulation of PDL1. Endogenous levels of miR-34a, -b, and -c in non–small cell lung cancer (NSCLC) cell lines. A) PDL1 expression in A549, H460, and H1299 cells transfected with miR-34a, -b, and -c. B-J) NSCLC cell lines were treated with miR-34a, miR-34b, or miR-34c at 100nM, and 24 hours later RNA was isolated to study miR-34a, -b, and -c transfection efficiency. At 96 hours after transfection, cell lysates were collected for protein analysis. Quantification of western blots shows that forced overexpression of miR-34a, miR-34b, or miR-34c suppressed PDL1 protein expression compared with a scrambled control. K) Luciferase activity in cells cotransfected with miR-34a, -b, or -c or a scrambled control and a luciferase reporter construct encoding the luciferase gene fused either to the wild-type PDL1 3’ UTR (PDL1 wt) or a mutated PDL1 3’ UTR (PDL1 mut). All three of the miR-34s reduced luciferase activity (P < .001, P =.006, and P =.006). An unpaired t test was used to calculate the two-sided P values. *P < .05, **P < .01, ***P < .001. Error bars on the bar charts represent standard deviation. Similar results were observed in three replicates. mut = mutated; NSCLC = non–small cell lung cancer; scr = scrambled; wt = wild-type.
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Figure 3: miR-34s regulation of PDL1. Endogenous levels of miR-34a, -b, and -c in non–small cell lung cancer (NSCLC) cell lines. A) PDL1 expression in A549, H460, and H1299 cells transfected with miR-34a, -b, and -c. B-J) NSCLC cell lines were treated with miR-34a, miR-34b, or miR-34c at 100nM, and 24 hours later RNA was isolated to study miR-34a, -b, and -c transfection efficiency. At 96 hours after transfection, cell lysates were collected for protein analysis. Quantification of western blots shows that forced overexpression of miR-34a, miR-34b, or miR-34c suppressed PDL1 protein expression compared with a scrambled control. K) Luciferase activity in cells cotransfected with miR-34a, -b, or -c or a scrambled control and a luciferase reporter construct encoding the luciferase gene fused either to the wild-type PDL1 3’ UTR (PDL1 wt) or a mutated PDL1 3’ UTR (PDL1 mut). All three of the miR-34s reduced luciferase activity (P < .001, P =.006, and P =.006). An unpaired t test was used to calculate the two-sided P values. *P < .05, **P < .01, ***P < .001. Error bars on the bar charts represent standard deviation. Similar results were observed in three replicates. mut = mutated; NSCLC = non–small cell lung cancer; scr = scrambled; wt = wild-type.
Mentions: The inverse correlation of miR-34 and PDL1 expression in NSCLC cells and in tumor samples implicated miR-34 as a regulator downstream of p53 to repress PDL1. This hypothesis was further corroborated by the observation that the 3’ untranslated region (UTR) of the PDL1 mRNA carries a putative miR-34 binding site at position 932–938 (32,33). We first analyzed endogenous levels of miR-34a, -b, and -c in NSCLC cell lines with different genetic backgrounds (Figure 3A). In agreement with our findings in NSCLC patient samples (Supplementary Figure 1, available online), miR-34a expression levels were higher than miR-34b and miR-34c levels in all three NSCLC cell lines. As shown in Figure 3, B-G, enforced overexpression of miR-34a suppressed the expression of PDL1 protein compared with a scrambled control. In addition, enforced overexpression of miR-34b or miR-34c suppressed the expression of PDL1 protein compared with a scrambled control (Figure 3, H-J). Next, to determine whether miR-34 interacts directly with the putative target gene PDL1, we cotransfected H1299 cells with miR-34a, -b, or -c mimics and a reporter vector encoding the luciferase gene that is fused to the 3′ UTR of the PDL1 gene (luc-PDL1). As shown in Figure 3K, luciferase activity was reduced in cells transfected with miR-34 and the luc-PDL1 construct compared with scrambled controls. In contrast, mutation of the predicted miR-34 binding site in the 3’ UTR of PDL1 rescued the luciferase activity, thus confirming that miR-34a, miR-34b, and miR-34c interact directly with the PDL1 3′ UTR (Figure 3K) (fold-change luciferase activity, miR-34a mean = 0.50, SD = 0.2, P < .001; miR-34b mean = 0.52, SD = 0.2, P = .006; and miR-34c mean = 0.59, SD = 0.14, P = .006).

Bottom Line: Finally, miR-34a delivery reduced the numbers of radiation-induced macrophages (mean of F4-80 expression percentage of control group = 52.4%, SD = 1.7%; mean of F4-80 expression percentage of MRX34 = 40.1%, SD = 3.5%, P = .008, n = 4) and T-regulatory cells.We identified a novel mechanism by which tumor immune evasion is regulated by p53/miR-34/PDL1 axis.Our results suggest that delivery of miRNAs with standard therapies, such as XRT, may represent a novel therapeutic approach for lung cancer.

View Article: PubMed Central - PubMed

Affiliation: Departments of Experimental Radiation Oncology (MAC, DV, XW, YY), Experimental Therapeutics (CI, GAC), and Radiation Oncology (RK, DRG, SK, JWW), The University of Texas MD Anderson Cancer Center, Houston, TX; Mirna Therapeutics, Inc., Austin, TX (HJP, KK, DM, AGB); Ohio State University, Columbus, OH (LA, DPC, KS); Texas Veterinary Pathology Associates (Houston), Houston, TX (DKG).

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Related in: MedlinePlus