Hsp90 … provide[s] at least two routes to the rapid evolution of new traits: (i) Acting as a potentiator, Hsp90’s folding reservoir allows individual genetic variation to immediately create new phenotypes; when the reservoir is compromised, the traits previously created by the potentiated variants disappear. (ii) Acting as a capacitor, Hsp90’s excess chaperone activity buffers the effects of other variants, storing them in a phenotypically silent form; when the Hsp90 reservoir is compromised, the effects of these variants are released, allowing them to create new traits. Jarosz and Lindquist (2010) [69
In 1958, Schabel suggested that model organisms such as yeast and bacteria can be used to understand drug resistance in cancer [125
]. For the past two decades, the Lindquist laboratory [69
], the Picard laboratory [126
], and other laboratories (e.g., [65
]), based on Schabel’s advice have used the yeast Saccharomyces cerevisiae
to understand how Hsp90 affects resistance or sensitivity. In a previous review [80
], we discussed how the Lindquist laboratory’s Hsp90-based drug-resistance studies might apply to drug resistance in cancer.
Natural variation Saccharomyces cerevisiae
can affect the growth rate of the yeast cells [69
]. Jarosz and Lindquist have reported that Hsp90 can act either as a “potentiator” or a “capacitor” for drug resistance and considered how this might affect the rapid evolution of new traits in general. Using recombinant inbred lines of bakers’ yeast (BY4716) and red wine yeast (RM11-1a) in the presence of anti-fungals, osmotic stressors, and other small molecules, they compared the growth rates in the presence or the absence of Hsp90 [69
]. Hsp90 was inhibited by the Hsp90 inhibitors radicicol and geldanamycin [69
]. Mechanistic models for the Hsp90-mediated potentiation or capacitation that may explain three of the findings described by Jarosz and Lindquist, rapamycin, hydroxyurea, and 1-chloro-2,4-nitrobenzine (CDNB), are shown in .
Hsp90 and drug resistance in yeast
The immunosuppressant rapamycin can prolong the life of mice [131
] and Drosophila [134
] and is also useful for treating breast and skin cancers [135
]. Jarosz and Lindquist found that BY4716 and RM11-1a yeast, and all recombinant inbred lines made from these two strains, have identical growth rates in the presence of Hsp90, but RM11-1a yeast have a ~3-fold increase in growth rate in the absence of Hsp90 compared with BY4716 [69
]. The recombinant inbred lines made from BY4716 and RM11-1a indicates that the NFS1
gene must have the RM11-1a genotype to confer rapamycin resistance (). Nfs1 protein is a cysteine desulfurase that acts as a sulfur donor in tRNA thiolation [139
], and yeast mutations in this same pathway confer rapamycin resistance [140
Jarosz and Lindquist [69
] have proposed that the Nfs1 protein is a client for Hsp90 and that Hsp90 folds the Nfs1 into a form that makes both RM11-1a and BY4716 yeast sensitive to rapamycin (, left). However, in the absence of Hsp90, Nfs1 with the RM11-1a genotype folds into a new conformation that is now resistant to rapamycin, but the BY4716 genotype protein remains in the rapamycin-sensitive conformation (, right). In other words, Hsp90 functions as a capacitor for the rapamycin resistant phenotype in the RM11-1a strain but not the BY4716 strain. In the absence of Hsp90, such as during stress, the previously hidden phenotype of rapamycin resistance is revealed by the new of the Nfs1-resistant (NFS1R
) conformation in the RM11-1a strain ().
Hydroxyurea is used to treat a variety of cancers, from leukemia to breast cancer [141
]. It is also used in combination with other drugs to treat head and neck cancer [145
]. One mechanism of action is thought to be through the inhibition of deoxyribonucleotide synthesis [146
]. Jarosz and Lindquist found that RM11-1a yeast are more resistant to hydroxyurea than BY4716 yeast in the presence of Hsp90, but that both BY4716 and RM11-1a yeast are resistant to hydroxyurea in the absence of Hsp90 [69
]. Analyses of the RM11-1a and BY4716 recombinant inbred lines indicate that the MEC1
gene from BY4716 confers the sensitivity to hydroxyurea (, left). Mec1 is a component of several checkpoint and DNA repair pathways in yeast [148
], and therefore likely repairs the DNA damage induced by hydroxyurea.
Jarosz and Lindquist [69
] further propose that Hsp90 functions as a capacitor in BY4716 yeast to make the Mec1 protein sensitive to hydroxyurea. However, according to their model, Hsp90 is not a chaperone for the Mec1 protein from RM11-1a yeast, but is a chaperone for Mec1 protein in BY4716 yeast (). In the absence of Hsp90, such as in stressful environments, the Mec1 protein in BY4716 yeast folds into a different conformation that is now more resistant to hydroxyurea (, right). Since the Mec1 protein in RM11-1a yeast is not a client for Hsp90, according to their model, it confers resistance to hydroxyurea regardless of whether Hsp90 is present or not (, right). This result is important because it suggests that what might be a client protein for Hsp90 in one genetic background might not be a client in another genetic background. If this is true in humans, which is likely, this would suggest a possible reason why Hsp90 inhibitors are more effective in some cancer patients than others when used in combination with other drugs ().
CDNB, a.k.a., DNCB (2,4-dinitro-1-chlorobenzine), is a redox cycling quinone that produces superoxide anions in its free radical state [152
]. Paper were published in the 1970s and 1980s [153
] that attempted to correlate skin-hypersensitivity caused by CDNB administration with cancer prognosis, with the concept of cancer being an autoimmune disease. We could not find any citations after 1987 in this regard. When exposed to CDNB, RM11-1a yeast show a remarkable 1500-fold increase in growth rate as compared to BY4716 yeast in the absence of Hsp90, and a 1500-fold increase in growth rate compared with both RM11-1a and BY4716 yeast in the presence of Hsp90 [69
]. This example is illustrative for two reasons, the first being the causative genetic polymorphism maps to the 3’ untranslated region of the NDI1
gene (, bottom left). The Ndi1 protein encodes an NADH-quinone (Q) oxidoreductase that protects against oxidative stress [158
]. CDNB produces oxidative stress both by directly producing free radicals, when in its free radical form, and by titrating GSH levels [161
]. Interestingly, overexpression of Ndi1 increases lifespan in Drosophila
], which is consistent with the free-radical theory of aging [171
]. The second reason is that it suggests that Hsp90 functions to regulate NDI1 expression in an indirect rather than a direct manner.
How might Hsp90 affect expression of NDI1 in RM11-1a yeast but not BY4716 yeast? We propose that Hsp90 is a chaperone for a hypothetical 3’UTR binding protein that binds to the NDI1 3’UTR when it has either the RM11-1a or the BY4716 genotype (, left). In the absence of Hsp90, according to our model, the 3’UTR binding protein folds into a different conformation (a circle) that no longer binds to the NDI1 3’UTR with the RM11-1a genotype, but it can still bind to the NDI1 3’UTR with the BY4716 genotype (, right). We propose that the 3’UTR binding protein is a translational repressor that also decreases the NDI1 mRNA levels when it is bound. Therefore, the NDI1 gene has much higher expression in RM11-1a yeast compared with BY4716 yeast (, right, thick arrow). This model would explain why CDNB resistance maps to the 3’UTR of the NDI1 gene and not the hypothetical 3’UTR binding protein.
A fascinating finding of Jarosz and Lindquist is that the clustering of the genotype and the phenotype in 11 different yeast strains is improved in the absence of Hsp90 [69
]. Genetic clustering was done by comparing the whole genome sequences of the 11 yeast strains. In the presence of Hsp90, there was no significant clustering of the phenotypes for resistance to 100 different growth conditions, including alternative carbon sources, oxidative stressors, antifungal drugs, small molecule drugs, and DNA damaging agents. However, in the absence of Hsp90, the phenotypes cluster as well as the genotypes. They conclude, “It is difficult to imagine how environmental stress in general, and Hsp90 in particular, could have such as strong impact on genotype-phenotype correlations unless it acted through the evolutionary history of these strains to influence the retention of a broad swath of genetic variation” [69
]. In other words, this is the best evidence to suggest that Hsp90 plays a critical role as a capacitor for phenotypic variation, such as in drug resistance in yeast, and probably also drug resistance in cancer. We predict that cancer cell phenotypes, such as growth rates in drug containing media, will cluster with the genotypes better when Hsp90 is inhibited. Understanding this relationship will be needed for facilitating personalized medicine approaches to treating cancer in humans with Hsp90 inhibitors used in combination with other drugs.