miRNAs are small non-coding RNAs able to modulate target-gene expression. It has been postulated that miRNAs confer robustness to biological processes, but a clear experimental evidence is still missing. Using a synthetic biology approach, we demonstrate that microRNAs provide phenotypic robustness to transcriptional regulatory networks by buffering fluctuations in protein levels. Here we construct a network motif in mammalian cells exhibiting a “toggle - switch” phenotype in which two alternative protein expression levels define its ON and OFF states. The motif consists of an inducible transcription factor that self-regulates its own transcription and that of a miRNA against the transcription factor itself. We confirm, using mathematical modeling and experimental approaches, that the microRNA confers robustness to the toggle-switch by enabling the cell to maintain and transmit its state. When absent, a dramatic increase in protein noise level occurs, causing the cell to randomly switch between the two states.

CD44 is a marker of cancer stem-like cells and epithelial-mesenchymal transition that is overexpressed in many cancer types, including thyroid carcinoma. At extracellular and intramembranous domains, CD44 undergoes sequential metalloprotease-and g-secretase-mediated proteolytic cleavage, releasing the intracellular protein fragment CD44-ICD, which translocates to the nucleus and activates gene transcription. Here, we show that CD44-ICD binds to the transcription factor CREB, increasing S133 phosphorylation and CREB-mediated gene transcription. CD44-ICD enhanced CREB recruitment to the cyclin D1 promoter, promoting cyclin D1 transcription and cell proliferation. Thyroid carcinoma cells harboring activated RET/PTC, RAS, or BRAF oncogenes exhibited CD44 cleavage and CD44-ICD accumulation. Chemical blockade of RET/PTC, BRAF, metalloprotease, or g-secretase were each sufficient to blunt CD44 processing. Furthermore, thyroid cancer cell proliferation was obstructed by RNA interference-mediated knockdown of CD44 or inhibition of g-secretase and adoptive CD44-ICD overexpression rescued cell proliferation. Together, these findings reveal a CD44-CREB signaling pathway that is needed to sustain cancer cell proliferation, potentially offering new molecular targets for therapeutic intervention in thyroid carcinoma. Cancer Res; 72(6);

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<p>PDF file- 142K, The RET-PTC-RAS-BRAF cascade triggers CD44 cleavage</p>

<p>PDF file - 175K, CD44-ICD-CREB complex in human PTC cell lines</p>

<p>PDF file - 102K, PKA and MEK inhibition blunts CD44-ICD mediated CREB phosphorylation</p>

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