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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* expression reduces tumor growth in vivo and direct intratumoral injection reduces tumor growth(A) Orthotopic xenografts of 22B cells expressing miR-27a* (pSuper-27a*) show reduced growth compared to control vector (pSuper); * at day 16 indicates point at which differences in tumor volume became statistically significant, p<0.05; (B) Tumor growth curve of 22B cells with inducible miR-27a* expression. One group of mice was treated with doxycycline (Doxy) to induce miR-27a* expression after tumors developed (day 0). Tumor volumes were significantly decreased in mice who received doxycycline; * marks day 18 when differences in tumor volumes became statistically significant, p<0.05; (C) Scatter plot depicting a statistically significant increase in miR-27a* RNA collected from murine tumor tissue in the doxycycline group compared to the non-doxycycline group, p<0.01; (D) Direct intratumoral injection significantly reduces growth of 17B tumors in mice treated with pSuper-27a* compared to control and pSuper; * marks day 18 when differences in tumor volumes became statistically significant (p<0.05) for pSuper-27a* compared to the control groups.
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Figure 5: miR-27a* expression reduces tumor growth in vivo and direct intratumoral injection reduces tumor growth(A) Orthotopic xenografts of 22B cells expressing miR-27a* (pSuper-27a*) show reduced growth compared to control vector (pSuper); * at day 16 indicates point at which differences in tumor volume became statistically significant, p<0.05; (B) Tumor growth curve of 22B cells with inducible miR-27a* expression. One group of mice was treated with doxycycline (Doxy) to induce miR-27a* expression after tumors developed (day 0). Tumor volumes were significantly decreased in mice who received doxycycline; * marks day 18 when differences in tumor volumes became statistically significant, p<0.05; (C) Scatter plot depicting a statistically significant increase in miR-27a* RNA collected from murine tumor tissue in the doxycycline group compared to the non-doxycycline group, p<0.01; (D) Direct intratumoral injection significantly reduces growth of 17B tumors in mice treated with pSuper-27a* compared to control and pSuper; * marks day 18 when differences in tumor volumes became statistically significant (p<0.05) for pSuper-27a* compared to the control groups.

Mentions: In order to assess the therapeutic potential of miR-27a* in a preclinical model, we created cell lines that stably express miR-27a*. 22B-pSuper-27a* constitutively expresses miR-27a* and decreases the expression of EGFR, AKT1, and mTOR. In a murine orthotopic xenograft model of oral cavity cancer, tumors from 22B-pSuper-27a* cells grew significantly slower than tumors from 22B-pSuper control cells (Fig. 5A). 22B-pSingle-27a* contains an inducible vector system resulting in miR-27a* expression when treated with doxycycline. As expected, the doxycycline-treated mice had significantly decreased tumor growth compared to the control group (Fig. 5B). Increased miR-27a* expression was confirmed by qRT-PCR in the tumors of the doxycycline-treated animals as compared to the control animals (Fig. 5C).


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* expression reduces tumor growth in vivo and direct intratumoral injection reduces tumor growth(A) Orthotopic xenografts of 22B cells expressing miR-27a* (pSuper-27a*) show reduced growth compared to control vector (pSuper); * at day 16 indicates point at which differences in tumor volume became statistically significant, p<0.05; (B) Tumor growth curve of 22B cells with inducible miR-27a* expression. One group of mice was treated with doxycycline (Doxy) to induce miR-27a* expression after tumors developed (day 0). Tumor volumes were significantly decreased in mice who received doxycycline; * marks day 18 when differences in tumor volumes became statistically significant, p<0.05; (C) Scatter plot depicting a statistically significant increase in miR-27a* RNA collected from murine tumor tissue in the doxycycline group compared to the non-doxycycline group, p<0.01; (D) Direct intratumoral injection significantly reduces growth of 17B tumors in mice treated with pSuper-27a* compared to control and pSuper; * marks day 18 when differences in tumor volumes became statistically significant (p<0.05) for pSuper-27a* compared to the control groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: miR-27a* expression reduces tumor growth in vivo and direct intratumoral injection reduces tumor growth(A) Orthotopic xenografts of 22B cells expressing miR-27a* (pSuper-27a*) show reduced growth compared to control vector (pSuper); * at day 16 indicates point at which differences in tumor volume became statistically significant, p<0.05; (B) Tumor growth curve of 22B cells with inducible miR-27a* expression. One group of mice was treated with doxycycline (Doxy) to induce miR-27a* expression after tumors developed (day 0). Tumor volumes were significantly decreased in mice who received doxycycline; * marks day 18 when differences in tumor volumes became statistically significant, p<0.05; (C) Scatter plot depicting a statistically significant increase in miR-27a* RNA collected from murine tumor tissue in the doxycycline group compared to the non-doxycycline group, p<0.01; (D) Direct intratumoral injection significantly reduces growth of 17B tumors in mice treated with pSuper-27a* compared to control and pSuper; * marks day 18 when differences in tumor volumes became statistically significant (p<0.05) for pSuper-27a* compared to the control groups.
Mentions: In order to assess the therapeutic potential of miR-27a* in a preclinical model, we created cell lines that stably express miR-27a*. 22B-pSuper-27a* constitutively expresses miR-27a* and decreases the expression of EGFR, AKT1, and mTOR. In a murine orthotopic xenograft model of oral cavity cancer, tumors from 22B-pSuper-27a* cells grew significantly slower than tumors from 22B-pSuper control cells (Fig. 5A). 22B-pSingle-27a* contains an inducible vector system resulting in miR-27a* expression when treated with doxycycline. As expected, the doxycycline-treated mice had significantly decreased tumor growth compared to the control group (Fig. 5B). Increased miR-27a* expression was confirmed by qRT-PCR in the tumors of the doxycycline-treated animals as compared to the control animals (Fig. 5C).

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