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miRs-134 and -370 function as tumor suppressors in colorectal cancer by independently suppressing EGFR and PI3K signalling.

El-Daly SM, Abba ML, Patil N, Allgayer H - Sci Rep (2016)

Bottom Line: Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis.Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins.Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Germany.

ABSTRACT
Growth factor receptor signalling plays a central and critical role in colorectal cancer. Most importantly, the EGFR signalling cascade involving PI3K/AKT/mTOR and Raf/MEK/ERK pathways are particularly relevant, since they are commonly activated in several cancer entities, including colorectal cancer. In this study, we show that miRs-134 and -370 are both capable of regulating these pathways by targeting EGFR and PIK3CA. In three different colorectal cancer cell lines (DLD1, HCT-116 and RKO), suppression of EGFR and PIK3CA through the enhanced expression of miR-134 or -370 led to a suppression of the key molecules of the PI3K/AKT/mTOR pathway. Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis. Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins. Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

No MeSH data available.


Related in: MedlinePlus

The PIK3CA/AKt/mTOR and Raf/MEK/ERK pathways are regulated by miRs-134 and-370.(a) Western blots showing validated targets and key molecules in the PIK3CA/AKT/mTOR and Raf/MEK/ERK signaling cascades following transfection with miR-134 or miR-370. (a) mimics; (b) Inhibitors, and (c) siRNAs in RKO, DLD1 and HCT-116 colorectal cancer cell lines. The probed proteins include EGFR, PIK3CA, AKT, p-Akt, p-c-Raf, p-mTOR, Rictor, ERK 1/2, p-ERK 1/2, MEK and p-MEK. Proteins were evaluated 48–72 hrs after transfection of cell lines, as indicated.
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f2: The PIK3CA/AKt/mTOR and Raf/MEK/ERK pathways are regulated by miRs-134 and-370.(a) Western blots showing validated targets and key molecules in the PIK3CA/AKT/mTOR and Raf/MEK/ERK signaling cascades following transfection with miR-134 or miR-370. (a) mimics; (b) Inhibitors, and (c) siRNAs in RKO, DLD1 and HCT-116 colorectal cancer cell lines. The probed proteins include EGFR, PIK3CA, AKT, p-Akt, p-c-Raf, p-mTOR, Rictor, ERK 1/2, p-ERK 1/2, MEK and p-MEK. Proteins were evaluated 48–72 hrs after transfection of cell lines, as indicated.

Mentions: Since EGFR and PIK3CA are both integral components of the PIK3CA/AKT and Raf/MEK/ERK pathways, we assessed the effect of miRs-134 and -370 not only on their individual targets, but also the entire signaling cascade. To evaluate this effect, we independently transfected the mimics and inhibitors of these two miRNAs in three colorectal cancer cell lines (RKO, DLD1 and HCT-116) and assessed protein expression and activitation of the key signaling molecules using Western blots with antibodies directed against whole-peptide and phosphorylated epitopes. Since miRNAs can target several genes simultaneously, we additionally transfected the same cell lines with validated siRNAs specific for the targets, to evaluate if observed changes were mediated, at least in part via EGFR or PIK3CA. For miR-134, we observed a suppression of EGFR and PIK3CA in all three cell lines with a subsequent repression of p-Akt, p-c-Raf, p-mTOR, Rictor and ERK1/2. AKT and MEK were not changed significantly in their protein amounts, however, p-ERK and p-MEK were elevated (Fig. 2a–c). Densitometry results for the Western Blots are provided in Supplementary Table 3). In the case of miR-370, the pattern seen for miR-134 was recapitulated, however not to the same degree as for miR-134. EGFR was not affected by miR-370 in the HCT-116 cell line, whereas significant repression was observed in RKO and DLD1 cell lines. Nonetheless, p-ERK and p-MEK were also elevated in all three cell lines (Fig. 2a–c). With the miRNA inhibitors, the reverse pattern to that of the mimics was observed in the key signaling molecules.


miRs-134 and -370 function as tumor suppressors in colorectal cancer by independently suppressing EGFR and PI3K signalling.

El-Daly SM, Abba ML, Patil N, Allgayer H - Sci Rep (2016)

The PIK3CA/AKt/mTOR and Raf/MEK/ERK pathways are regulated by miRs-134 and-370.(a) Western blots showing validated targets and key molecules in the PIK3CA/AKT/mTOR and Raf/MEK/ERK signaling cascades following transfection with miR-134 or miR-370. (a) mimics; (b) Inhibitors, and (c) siRNAs in RKO, DLD1 and HCT-116 colorectal cancer cell lines. The probed proteins include EGFR, PIK3CA, AKT, p-Akt, p-c-Raf, p-mTOR, Rictor, ERK 1/2, p-ERK 1/2, MEK and p-MEK. Proteins were evaluated 48–72 hrs after transfection of cell lines, as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837379&req=5

f2: The PIK3CA/AKt/mTOR and Raf/MEK/ERK pathways are regulated by miRs-134 and-370.(a) Western blots showing validated targets and key molecules in the PIK3CA/AKT/mTOR and Raf/MEK/ERK signaling cascades following transfection with miR-134 or miR-370. (a) mimics; (b) Inhibitors, and (c) siRNAs in RKO, DLD1 and HCT-116 colorectal cancer cell lines. The probed proteins include EGFR, PIK3CA, AKT, p-Akt, p-c-Raf, p-mTOR, Rictor, ERK 1/2, p-ERK 1/2, MEK and p-MEK. Proteins were evaluated 48–72 hrs after transfection of cell lines, as indicated.
Mentions: Since EGFR and PIK3CA are both integral components of the PIK3CA/AKT and Raf/MEK/ERK pathways, we assessed the effect of miRs-134 and -370 not only on their individual targets, but also the entire signaling cascade. To evaluate this effect, we independently transfected the mimics and inhibitors of these two miRNAs in three colorectal cancer cell lines (RKO, DLD1 and HCT-116) and assessed protein expression and activitation of the key signaling molecules using Western blots with antibodies directed against whole-peptide and phosphorylated epitopes. Since miRNAs can target several genes simultaneously, we additionally transfected the same cell lines with validated siRNAs specific for the targets, to evaluate if observed changes were mediated, at least in part via EGFR or PIK3CA. For miR-134, we observed a suppression of EGFR and PIK3CA in all three cell lines with a subsequent repression of p-Akt, p-c-Raf, p-mTOR, Rictor and ERK1/2. AKT and MEK were not changed significantly in their protein amounts, however, p-ERK and p-MEK were elevated (Fig. 2a–c). Densitometry results for the Western Blots are provided in Supplementary Table 3). In the case of miR-370, the pattern seen for miR-134 was recapitulated, however not to the same degree as for miR-134. EGFR was not affected by miR-370 in the HCT-116 cell line, whereas significant repression was observed in RKO and DLD1 cell lines. Nonetheless, p-ERK and p-MEK were also elevated in all three cell lines (Fig. 2a–c). With the miRNA inhibitors, the reverse pattern to that of the mimics was observed in the key signaling molecules.

Bottom Line: Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis.Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins.Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Germany.

ABSTRACT
Growth factor receptor signalling plays a central and critical role in colorectal cancer. Most importantly, the EGFR signalling cascade involving PI3K/AKT/mTOR and Raf/MEK/ERK pathways are particularly relevant, since they are commonly activated in several cancer entities, including colorectal cancer. In this study, we show that miRs-134 and -370 are both capable of regulating these pathways by targeting EGFR and PIK3CA. In three different colorectal cancer cell lines (DLD1, HCT-116 and RKO), suppression of EGFR and PIK3CA through the enhanced expression of miR-134 or -370 led to a suppression of the key molecules of the PI3K/AKT/mTOR pathway. Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis. Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins. Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

No MeSH data available.


Related in: MedlinePlus