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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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Overexpression of EGFR axis signaling components reverses the loss of HNSCC cell viability mediated by miR-27a*, which increases apoptosis and reduces migration(A) The effect of miR-27a* on cell viability is decreased in 22A and 22B cells by overexpression of EGFR, AKT1 and mTOR 48 hrs prior to miR-27a* expression as compared to control vector, *p<0.01; (B) Overexpression following transfection with EGFR, AKT1 and mTOR (EAmT) vectors was confirmed by immunoblot analysis. (C) Annexin V assay shows increased apoptosis in HNSCC cells after transfection with miR-27a*, *p<0.005; (D) Transfection with miR-27a* leads to increased PARP cleavage compared to miR-27a and -Control; (E) Wound healing assay demonstrates decreased migration distance after 24 hrs after transfection of miR-27a* compared to miR-Control, −27a, and −7, *p<0.001.
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Figure 4: Overexpression of EGFR axis signaling components reverses the loss of HNSCC cell viability mediated by miR-27a*, which increases apoptosis and reduces migration(A) The effect of miR-27a* on cell viability is decreased in 22A and 22B cells by overexpression of EGFR, AKT1 and mTOR 48 hrs prior to miR-27a* expression as compared to control vector, *p<0.01; (B) Overexpression following transfection with EGFR, AKT1 and mTOR (EAmT) vectors was confirmed by immunoblot analysis. (C) Annexin V assay shows increased apoptosis in HNSCC cells after transfection with miR-27a*, *p<0.005; (D) Transfection with miR-27a* leads to increased PARP cleavage compared to miR-27a and -Control; (E) Wound healing assay demonstrates decreased migration distance after 24 hrs after transfection of miR-27a* compared to miR-Control, −27a, and −7, *p<0.001.

Mentions: Although miR-27a* targets EGFR, AKT1 and mTOR directly and independently within the EGFR signaling pathway, the specific effect of those regulatory events on the overall decreased viability of HNSCC cells has not been established. EGFR, AKT1 and mTOR CDS expression vectors were transfected into HNSCC cells prior to the introduction of miR-27a*. Overexpression of EGFR, AKT1 and mTOR decreased the effect of miR-27a* expression on HNSCC cell viability as compared to control vector alone (Fig. 4A). However, expression of these EGFR signaling pathway components did not completely abrogate the effect of miR-27a* on HNSCC cell viability. We confirmed increased expression of EGFR, AKT1 and mTOR by immunoblotting analysis (Fig. 4B). Thus, these findings suggest that the effect of miR-27a* expression on HNSCC cell viability was related, at least in part, to the EGFR signaling pathway.


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)

Overexpression of EGFR axis signaling components reverses the loss of HNSCC cell viability mediated by miR-27a*, which increases apoptosis and reduces migration(A) The effect of miR-27a* on cell viability is decreased in 22A and 22B cells by overexpression of EGFR, AKT1 and mTOR 48 hrs prior to miR-27a* expression as compared to control vector, *p<0.01; (B) Overexpression following transfection with EGFR, AKT1 and mTOR (EAmT) vectors was confirmed by immunoblot analysis. (C) Annexin V assay shows increased apoptosis in HNSCC cells after transfection with miR-27a*, *p<0.005; (D) Transfection with miR-27a* leads to increased PARP cleavage compared to miR-27a and -Control; (E) Wound healing assay demonstrates decreased migration distance after 24 hrs after transfection of miR-27a* compared to miR-Control, −27a, and −7, *p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Overexpression of EGFR axis signaling components reverses the loss of HNSCC cell viability mediated by miR-27a*, which increases apoptosis and reduces migration(A) The effect of miR-27a* on cell viability is decreased in 22A and 22B cells by overexpression of EGFR, AKT1 and mTOR 48 hrs prior to miR-27a* expression as compared to control vector, *p<0.01; (B) Overexpression following transfection with EGFR, AKT1 and mTOR (EAmT) vectors was confirmed by immunoblot analysis. (C) Annexin V assay shows increased apoptosis in HNSCC cells after transfection with miR-27a*, *p<0.005; (D) Transfection with miR-27a* leads to increased PARP cleavage compared to miR-27a and -Control; (E) Wound healing assay demonstrates decreased migration distance after 24 hrs after transfection of miR-27a* compared to miR-Control, −27a, and −7, *p<0.001.
Mentions: Although miR-27a* targets EGFR, AKT1 and mTOR directly and independently within the EGFR signaling pathway, the specific effect of those regulatory events on the overall decreased viability of HNSCC cells has not been established. EGFR, AKT1 and mTOR CDS expression vectors were transfected into HNSCC cells prior to the introduction of miR-27a*. Overexpression of EGFR, AKT1 and mTOR decreased the effect of miR-27a* expression on HNSCC cell viability as compared to control vector alone (Fig. 4A). However, expression of these EGFR signaling pathway components did not completely abrogate the effect of miR-27a* on HNSCC cell viability. We confirmed increased expression of EGFR, AKT1 and mTOR by immunoblotting analysis (Fig. 4B). Thus, these findings suggest that the effect of miR-27a* expression on HNSCC cell viability was related, at least in part, to the EGFR signaling pathway.

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