Enhanced squalene production by modulation of pathways consuming squalene and its precursor.

Fermentative production of squalene in yeast as an alternative approach to extracting squalene from sharks or plants has attracted significant interest. However, squalene accumulation is limited due to its inevitable high-flux allocation toward ergosterol synthesis. In this study, we described expression control of squalene monooxygenase (Erg1p), the first-step enzyme of ergosterol synthesis from squalene, to significantly reduce squalene loss. We replaced the ERG1 promoter (PERG1) with three natural yeast promoters with different activities (PPCL2, PHCM1, and PTHI2). ERG1 controlled by PTHI2 showed 20 times higher squalene production compared with the wild-type strain, whereas the other two strains exhibited no significant difference. By combining the overexpression of rate-limiting enzyme and the deletion of non-essential competing pathway gene, the yeast Saccharomyces cerevisiae produced up to 379 mg/L of squalene.

MMP24 as a Target of YAP is a Potential Prognostic Factor in Cancer Patients.

The extracellular matrix (ECM) surrounding cancer cells becomes stiffer during tumor progression, which influences cancer cell behaviors such as invasion and proliferation through modulation of gene expression as well as remodeling of the actin cytoskeleton. In this study, we show that MMP24 encoding matrix metalloproteinase (MMP)-24 is a novel target gene of Yes-associated protein (YAP), a transcription coactivator known as a mechanotransducer. We first examined the effect of substrate stiffness on MMP24 expression in MCF-7 human breast cancer cells and showed that the expression of MMP24 was significantly higher in cells grown on stiff substrates than that on soft substrates. The MMP24 expression was significantly reduced by knockdown of YAP. In contrast, the expression of constitutively active YAP increased MMP24 promoter activity. In addition, binding of YAP to the MMP24 promoter was confirmed by the chromatin immunoprecipitation (ChIP) assay. These results show that ECM stiffening promotes YAP activation, thereby inducing MMP24 expression. Based on the Human Protein Atlas database, breast cancer patients with lower MMP24 expression exhibit the worse survival rates overall. Thus, MMP24 may negatively regulate the aggressiveness of cancer cells under the stiff ECM environment during tumor progression.

Substrate rigidity-dependent positive feedback regulation between YAP and ROCK2.

Extracellular matrix (ECM) stiffness influences gene expression, leading to modulation of various cellular functions. While ROCK2 regulates actomyosin activity as well as cell migration and proliferation, expression of ROCK2 is increased in response to stiffening ECM. However, the mechanism underlying rigidity-dependent ROCK2 expression remains elusive. Here, we show that YAP, a mechanically regulated transcription coactivator, upregulates ROCK2 expression in an ECM rigidity-dependent manner. YAP interacted with the ROCK2 promoter region in an actomyosin activity-dependent manner. Knockdown of YAP decreased ROCK2 expression while activity of the ROCK2 promoter was upregulated by expressing constitutively active YAP. Furthermore, we found that ROCK2 expression promotes transcriptional activation by YAP. Our results reveal a novel positive feedback loop between YAP and ROCK2, which is modulated by ECM stiffness.

Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression.

The physical properties of the extracellular matrix (ECM), such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK) 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.