Tumor-intrinsic and tumor-extrinsic factors impacting hsp90- targeted therapy.

In 1994 the first heat shock protein 90 (Hsp90) inhibitor was identified and Hsp90 was reported to be a target for anticancer therapeutics. In the past 18 years there have been 17 distinct Hsp90 inhibitors entered into clinical trial, and the small molecule Hsp90 inhibitors have been highly valuable as probes of the role of Hsp90 and its client proteins in cancer. Although no Hsp90 inhibitor has achieved regulatory approval, recently there has been significant progress in Hsp90 inhibitor clinical development, and in the past year RECIST responses have been documented in HER2-positive breast cancer and EML4-ALK-positive non-small cell lung cancer. All of the clinical Hsp90 inhibitors studied to date are specific in their target, i.e. they bind exclusively to Hsp90 and two related heat shock proteins. However, Hsp90 inhibitors are markedly pleiotropic, causing degradation of over 200 client proteins and impacting critical multiprotein complexes. Furthermore, it has only recently been appreciated that Hsp90 inhibitors can, paradoxically, cause transient activation of the protein kinase clients they are chaperoning, resulting in initiation of signal transduction and significant physiological events in both tumor and tumor microenvironment. An additional area of recent progress in Hsp90 research is in studies of the posttranslational modifications of Hsp90 itself and Hsp90 co-chaperone proteins. Together, a picture is emerging in which the impact of Hsp90 inhibitors is shaped by the tumor intracellular and extracellular milieu, and in which Hsp90 inhibitors impact tumor and host on a microenvironmental and systems level. Here we review the tumor intrinsic and extrinsic factors that impact the efficacy of small molecules engaging the Hsp90 chaperone machine.

The ZbYME2 gene from the food spoilage yeast Zygosaccharomyces bailii confers not only YME2 functions in Saccharomyces cerevisiae, but also the capacity for catabolism of sorbate and benzoate, two major weak organic acid preservatives.

A factor influencing resistances of food spoilage microbes to sorbate and benzoate is whether these organisms are able to catalyse the degradation of these preservative compounds. Several fungi metabolize benzoic acid by the beta-ketoadipate pathway, involving the hydroxylation of benzoate to 4-hydroxybenzoate. Saccharomyces cerevisiae is unable to use benzoate as a sole carbon source, apparently through the lack of benzoate-4-hydroxylase activity. However a single gene from the food spoilage yeast Zygosaccharomyces bailii, heterologously expressed in S. cerevisiae cells, can enable growth of the latter on benzoate, sorbate and phenylalanine. Although this ZbYME2 gene is essential for benzoate utilization by Z. bailii, its ZbYme2p product has little homology to other fungal benzoate-4-hydroxylases studied to date, all of which appear to be microsomal cytochrome P450s. Instead, ZbYme2p has strong similarity to the matrix domain of the S. cerevisiae mitochondrial protein Yme2p/Rna12p/Prp12p and, when expressed as a functional fusion to green fluorescent protein in S. cerevisiae growing on benzoate, is largely localized to mitochondria. The phenotypes associated with loss of the native Yme2p from S. cerevisiae, mostly apparent in yme1,yme2 cells, may relate to increased detrimental effects of endogenous oxidative stress. Heterologous expression of ZbYME2 complements these phenotypes, yet it also confers a potential for weak acid preservative catabolism that the native S. cerevisiae Yme2p is unable to provide. Benzoate utilization by S. cerevisiae expressing ZbYME2 requires a functional mitochondrial respiratory chain, but not the native Yme1p and Yme2p of the mitochondrion.

Targeted gene deletion in Zygosaccharomyces bailii.

Yeasts of the genus Zygosaccharomyces are notable agents of large-scale food spoilage. Despite the economic importance of these organisms, little is known about the stress adaptations whereby they adapt to many of the more severe conditions of food preservation. In this study it was shown that genes of Z. bailii, a yeast notable for its high resistances to food preservatives and ethanol, can be isolated by complementation of the corresponding mutant strains of Saccharomyces cerevisiae. It was also discovered that the acquisition by S. cerevisiae of a single small Z. bailii gene (ZbYME2) was sufficient for the former yeast to acquire the ability to degrade two major food preservatives, benzoic acid and sorbic acid. Using DNA cassettes containing dominant selectable markers and methods originally developed for performing gene deletions in S. cerevisiae, the two copies of ZbYME2 in the Z. bailii genome were sequentially deleted. The resulting Zbyme2/Zbyme2 homozygous deletant strain had lost any ability to utilize benzoate as sole carbon source and was more sensitive to weak acid preservatives during growth on glucose. Thus, ZbYME2, probably the nuclear gene for a mitochondrial mono-oxygenase function, is essential for Z. bailii to degrade food preservatives. This ability to catabolize weak acid preservatives is a significant factor contributing to the preservative resistance of Z. bailii under aerobic conditions. This study is the first to demonstrate that it is possible to delete in Z. bailii genes that are suspected as being important for growth of this organism in preserved foods and beverages. With the construction of further mutant of Z. bailii strains, a clearer picture should emerge of how this yeast adapts to the conditions of food preservation. This information will, in turn, allow the design of new preservation strategies. GenBank Accession Nos: ZbURA3 (AF279259), ZbTIM9 (AF279260), ZbYME2 (AF279261), ZbTRP1 (AF279262), ZbHHT1(AF296170).