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Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Wu X, Bhayani MK, Dodge CT, Nicoloso MS, Chen Y, Yan X, Adachi M, Thomas L, Galer CE, Jiffar T, Pickering CR, Kupferman ME, Myers JN, Calin GA, Lai SY - Oncotarget (2013)

Bottom Line: Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a.Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth.Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

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miR-27a* coordinately downregulates the EGFR signaling axis via independent direct interactions with EGFR, AKT1, and mTOR(A) Further in silico screening of downstream members of the EGFR signaling axis, identified 5 putative binding sites for miR-27a* (black bars) on AKT1 and 11 binding sites for miR-27a* on mTOR; (B) Immunoblot shows decreased EGFR expression after the transfection of miR-27a*, −27a and −7 precursors. Downstream AKT1 and mTOR are also decreased when transfected with miR-27a*, but not miR-27a or −7; (C) Densitometry analysis quantifies the differences observed in the immunoblots, *p<0.01; (D) Detailed map of reporter plasmid constructs depicting the EGFR sequence placed downstream of luciferase in pGL3; (E) Luciferase assay after transfection of EGFR reporter plasmids and miR-27a* in HNSCC cells demonstrates E2145-pGL3 and E3908-pGL3 have functional binding sites for miR-27a* and that E160-pGL3 is a non-functional site, *p<0.05 and **p<0.005; (F) Luciferase assay after transfection of AKT1 and mTOR reporter plasmids plus miR-27a* in HNSCC cells demonstrates the 3'UTR of both signaling mediators have functional binding sites for miR-27a*, *p<0.001.
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Figure 3: miR-27a* coordinately downregulates the EGFR signaling axis via independent direct interactions with EGFR, AKT1, and mTOR(A) Further in silico screening of downstream members of the EGFR signaling axis, identified 5 putative binding sites for miR-27a* (black bars) on AKT1 and 11 binding sites for miR-27a* on mTOR; (B) Immunoblot shows decreased EGFR expression after the transfection of miR-27a*, −27a and −7 precursors. Downstream AKT1 and mTOR are also decreased when transfected with miR-27a*, but not miR-27a or −7; (C) Densitometry analysis quantifies the differences observed in the immunoblots, *p<0.01; (D) Detailed map of reporter plasmid constructs depicting the EGFR sequence placed downstream of luciferase in pGL3; (E) Luciferase assay after transfection of EGFR reporter plasmids and miR-27a* in HNSCC cells demonstrates E2145-pGL3 and E3908-pGL3 have functional binding sites for miR-27a* and that E160-pGL3 is a non-functional site, *p<0.05 and **p<0.005; (F) Luciferase assay after transfection of AKT1 and mTOR reporter plasmids plus miR-27a* in HNSCC cells demonstrates the 3'UTR of both signaling mediators have functional binding sites for miR-27a*, *p<0.001.

Mentions: Given that miR-7 has known interactions with EGFR as both a tumor suppressor and oncogene [25, 29], but did not show an effect on cell viability in HNSCC, we postulated that miR-27a* targets additional genes in the EGFR signaling axis to reduce cell survival. Further in silico analysis identified AKT1 and mTOR as additional targets of miR-27a* (Fig. 3A). These findings were confirmed with analysis of protein expression in HNSCC cells transfected with miR-27a*, resulting in reduced protein levels of EGFR, AKT1 and mTOR compared to miR-control (Control). In contrast, miR-27a and −7 decreased EGFR expression without modulation of AKT1 or mTOR (Figs. 3B and 3C). Moreover, we confirmed the effect of miR-27a* on mRNA levels by qRT-PCR (Fig. S3A). Transfection of HNSCC cells with miR-27a*-IH slightly increased EGFR, AKT1 and mTOR expression as compared to miR-27a* and -Control (Fig. S3B). In HOK16B, EGFR expression was decreased by miR-27a* and slightly increased by miR-27a*-IH. Taken together, these results demonstrate post-transcriptional regulation of EGFR, AKT1, and mTOR by miR-27a*.


Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Wu X, Bhayani MK, Dodge CT, Nicoloso MS, Chen Y, Yan X, Adachi M, Thomas L, Galer CE, Jiffar T, Pickering CR, Kupferman ME, Myers JN, Calin GA, Lai SY - Oncotarget (2013)

miR-27a* coordinately downregulates the EGFR signaling axis via independent direct interactions with EGFR, AKT1, and mTOR(A) Further in silico screening of downstream members of the EGFR signaling axis, identified 5 putative binding sites for miR-27a* (black bars) on AKT1 and 11 binding sites for miR-27a* on mTOR; (B) Immunoblot shows decreased EGFR expression after the transfection of miR-27a*, −27a and −7 precursors. Downstream AKT1 and mTOR are also decreased when transfected with miR-27a*, but not miR-27a or −7; (C) Densitometry analysis quantifies the differences observed in the immunoblots, *p<0.01; (D) Detailed map of reporter plasmid constructs depicting the EGFR sequence placed downstream of luciferase in pGL3; (E) Luciferase assay after transfection of EGFR reporter plasmids and miR-27a* in HNSCC cells demonstrates E2145-pGL3 and E3908-pGL3 have functional binding sites for miR-27a* and that E160-pGL3 is a non-functional site, *p<0.05 and **p<0.005; (F) Luciferase assay after transfection of AKT1 and mTOR reporter plasmids plus miR-27a* in HNSCC cells demonstrates the 3'UTR of both signaling mediators have functional binding sites for miR-27a*, *p<0.001.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824521&req=5

Figure 3: miR-27a* coordinately downregulates the EGFR signaling axis via independent direct interactions with EGFR, AKT1, and mTOR(A) Further in silico screening of downstream members of the EGFR signaling axis, identified 5 putative binding sites for miR-27a* (black bars) on AKT1 and 11 binding sites for miR-27a* on mTOR; (B) Immunoblot shows decreased EGFR expression after the transfection of miR-27a*, −27a and −7 precursors. Downstream AKT1 and mTOR are also decreased when transfected with miR-27a*, but not miR-27a or −7; (C) Densitometry analysis quantifies the differences observed in the immunoblots, *p<0.01; (D) Detailed map of reporter plasmid constructs depicting the EGFR sequence placed downstream of luciferase in pGL3; (E) Luciferase assay after transfection of EGFR reporter plasmids and miR-27a* in HNSCC cells demonstrates E2145-pGL3 and E3908-pGL3 have functional binding sites for miR-27a* and that E160-pGL3 is a non-functional site, *p<0.05 and **p<0.005; (F) Luciferase assay after transfection of AKT1 and mTOR reporter plasmids plus miR-27a* in HNSCC cells demonstrates the 3'UTR of both signaling mediators have functional binding sites for miR-27a*, *p<0.001.
Mentions: Given that miR-7 has known interactions with EGFR as both a tumor suppressor and oncogene [25, 29], but did not show an effect on cell viability in HNSCC, we postulated that miR-27a* targets additional genes in the EGFR signaling axis to reduce cell survival. Further in silico analysis identified AKT1 and mTOR as additional targets of miR-27a* (Fig. 3A). These findings were confirmed with analysis of protein expression in HNSCC cells transfected with miR-27a*, resulting in reduced protein levels of EGFR, AKT1 and mTOR compared to miR-control (Control). In contrast, miR-27a and −7 decreased EGFR expression without modulation of AKT1 or mTOR (Figs. 3B and 3C). Moreover, we confirmed the effect of miR-27a* on mRNA levels by qRT-PCR (Fig. S3A). Transfection of HNSCC cells with miR-27a*-IH slightly increased EGFR, AKT1 and mTOR expression as compared to miR-27a* and -Control (Fig. S3B). In HOK16B, EGFR expression was decreased by miR-27a* and slightly increased by miR-27a*-IH. Taken together, these results demonstrate post-transcriptional regulation of EGFR, AKT1, and mTOR by miR-27a*.

Bottom Line: Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a.Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth.Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

ABSTRACT
Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.

Show MeSH
Related in: MedlinePlus