We have determined that many genes affected by p23 overexpression in MCF-7 cells are also misregulated in advanced breast cancers. These genes display varying sensitivities to treatments with Hsp90 inhibitors, TAM, or E2, which could play a role in the responsiveness of tumor cells to anti-estrogen treatment or chemotherapy. For example, ABCC3 is upregulated by p23 overexpression alone, is Hsp90-dependent, further upregulated by TAM treatment, and downregulated by E2 treatment. Functionally, ABCC3 confers selective resistance to chemotherapeutic agents in MCF-7+p23 cells when compared to control cells, and we have observed preliminarily that siRNA-mediated or pharmacological depletion of ER, using ICI treatment, decreases the sensitivity of MCF-7 control cells to doxorubicin (data not shown). Thus, both gene expression data and functional studies demonstrate that p23 via ABCC3 may regulate chemotherapeutic resistance acquired in metastatic and recurrent ER-negative breast tumors.
How might p23 selectively affect gene expression? p23 has been suggested to play a role in the binding and clearance of steroid receptors at response elements (8
). Our results also indicate that p23 increases recruitment of ER to EREs of E2-repressed genes ABCC3 and PMP22 () as well as to ER-activated genes pS2 and cathepsin D (10
). Thus, certain transcription factors, like steroid receptors, may utilize p23 to efficiently cycle on and off DNA to modulate gene transcription.
We find that “active” histone modifications (H3-AcK9/14) are present at the promoters of p23-sensitive genes (). The fact that these modifications are also present at the promoters of some p23-sensitive genes in MCF-7 control cells where p23 is not overexpressed () indicate that p23 may not need to overcome the transcriptional barriers associated with gene silencing in order to upregulate gene expression. Therefore, a mechanism p23 may use to increase gene expression could be to recruit HATs implicated in transcription re-initiation (i.e. GCN5), rather than those involved in transcription initiation (i.e. p300) (45
) to raise the transcriptional output from already “active” promoters.
Indeed, we have observed a functional interaction between p23 and the HAT GCN5 by conducting a synthetic lethal screen in yeast, suggesting that p23 affects the activity of HAT pathways in an overlapping fashion with GCN5 (Oxelmark and Garabedian, unpublished). Histone H3-K9/14 is a target for acetylation by GCN5 (39
), which further implicates GCN5 as a HAT mediating hyperaceylation at the promoters of p23-upregulated genes. Interestingly, two metabolic genes regulated by p23, ACSL1 and KYNU, which are upregulated in metastatic and recurrent breast cancers, are also known to regulate the activity of HATs and PARP chromatin remodeling enzymes (39
). Therefore, a plausible model for p23-sensitive gene regulation is that p23 may influence the DNA binding dynamics of specific transcription factors to up- or downregulate gene expression. p23 may also modulate the recruitment of co-activators necessary to re-initiate transcription from active loci to increase gene expression. Further, amplification of gene regulation in situations of p23 overexpression may occur through the enhancement of p23-sensitive metabolic pathways that affect the activity of chromatin modifying enzymes. Whether or not p23 is recruiting these factors directly is still under investigation.
p23 localizes to both the nucleus and cytoplasm in MCF-7 cells (Figure S7
) and high cytoplasmic p23 correlates with shorter disease-free survival times for breast cancer patients, indicating that p23 can promote tumorigenesis by functioning in multiple cellular compartments. In fact, we observe increased activated Akt1 in the cytoplasm and an expanded phosphoproteome in the cytoplasm and nucleus of MCF-7+p23 compared to control cells, suggesting that p23 overexpression facilitates, either directly or indirectly, enhanced kinase activity and protein phosphorylation (Figure S8
). Given that activated Akt is a potent survival signal, our findings also suggest that tumors overexpressing p23 would have a survival advantage (48
). Therefore, we propose that cancers with high levels of p23 activate signaling pathways in the cytoplasm, which can also amplify p23-sensitive gene transcription in the nucleus.
Our findings strongly suggest that individuals with breast cancers that display high p23 protein levels are more likely to exhibit lymph node involvement and experience disease recurrence and mortality. This is consistent with our in vitro
model that p23 overexpression elicits gene expression and phenotypic changes in MCF-7 cells akin to those that occur as breast tumors become invasive. Given that nodal status is the most significant factor in predicting survival (49
), it will be important to find ways to reduce p23 expression to decrease lymph node metastases. Lowering p23 expression should also mitigate p23-mediated chemotherapeutic resistance (etoposide, doxorubicin). Recently, celastrol, the active compound in “Thunder of God Vine” root extracts used in traditional Chinese medicine, has been shown to bind and inactivate p23 by triggering its oligomerization into fibers (50
). Thus, targeting p23 should reduce invasiveness and drug resistance in breast cancer cells and prevent disease recurrence.