Metastatic breast cancer remains challenging to treat, and most patients ultimately progress on therapy. This acquired drug resistance is largely due to drug-refractory sub-populations (subclones) within heterogeneous tumors. Here, we track the genetic and phenotypic subclonal evolution of four breast cancers through years of treatment to better understand how breast cancers become drug-resistant. Recurrently appearing post-chemotherapy mutations are rare. However, bulk and single-cell RNA sequencing reveal acquisition of malignant phenotypes after treatment, including enhanced mesenchymal and growth factor signaling, which may promote drug resistance, and decreased antigen presentation and TNF-α signaling, which may enable immune system avoidance. Some of these phenotypes pre-exist in pre-treatment subclones that become dominant after chemotherapy, indicating selection for resistance phenotypes. Post-chemotherapy cancer cells are effectively treated with drugs targeting acquired phenotypes. These findings highlight cancer’s ability to evolve phenotypically and suggest a phenotype-targeted treatment strategy that adapts to cancer as it evolves.

Tumor suppressor p53 exhibits an enigmatic phenotype in cells exposed to electrophilic, cyclopentenone prostaglandins of the A and J series. Namely, cells harboring a wild-type p53 gene accumulate p53 protein that is conformationally and functionally impaired. This occurs via an unknown molecular mechanism. We report that electrophilic cyclopentenone prostaglandins covalently modify and inhibit thioredoxin reductase, a selenoprotein that governs p53 and other redox-sensitive transcription factors. This mechanism accounts fully for the unusual p53 phenotype in cells exposed to electrophilic prostaglandins. Based on this mechanism we derived, tested, and affirmed several predictions regarding the kinetics of p53 inactivation; the protective effects of selenium; the structure-activity relationships for inhibition of thioredoxin reductase and impairment of p53 by electrophilic lipids; the susceptibility of hypoxia-inducible factor to inactivation by electrophilic lipids; and the equivalence of chemical inactivation of p53 to deletion of a p53 allele. Chemical precepts dictate that other electrophilic agents should also inhibit thioredoxin reductase and impair its governance of redoxsensitive proteins. Our results provide a novel framework to understand how endogenous and exogenous electrophiles might participate in carcinogenesis; how selenoproteins and selenium might confer protection against cancer; how certain tumors might acquire their paradoxical p53 phenotype; and how chronic inflammation might heighten the risk for cancer.

There is ongoing concern regarding the toxicity of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. Two commercial ZnO types, one sold as a 8-10 nm powder and the other described as -325 mesh (<44 mum) powder, were evaluated in human colon-derived RKO cells. The powders had a volume-to-surface area ratio equivalent to 40 and 330 nm spheres, respectively. Both materials formed micrometer-sized agglomerates in cell culture media. The nanosized ZnO was more cytotoxic than the micrometer-sized ZnO with LC(50) values of 15 +/- 1 and 29 +/- 4 mug/cm(2), respectively. Transfer of Zn from the solid phase to the cell culture media in the presence of RKO cells was time- and concentration-dependent. However, direct particle-cell contact was required for RKO cell cytotoxicity, and the toxicity of particles was independent of the amount of soluble Zn in the cell culture media. The mechanism of cell death includes the disruption of mitochondrial function. Robust markers of apoptosis, Annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were observed when cells were treated with ZnO particulate matter but not when treated with comparable concentration of a soluble Zn salt. Both ZnO samples induced similar mechanisms of toxicity, but there was a statistically significant increase in potency per unit mass with the smaller particles.

Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated ␣,␤-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic ␣,␤-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by ⌬12-prostaglandin J 2 . This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents. Certain electrophilic prostaglandins (PG)1 can repress transactivation by NFB and p53 (1-4), two prominent transcription factors that govern the decision of a cell to survive or die (5-7). This transcriptional repression is a pharmacologically unique trait that distinguishes PG of the A and J series from other PG that act via membrane-spanning receptors (8). If the endocyclic ␣,␤-unsaturated ketone shared by A and J series PG confers activity, as hypothesized (1, 2, 4, 9, 10), then two predictions should be valid. First, individual A series and J series PG should act rather uniformly on the cellular processes they affect. Second, their cellular effects should be self-consistent with established models of NFB and p53 function. However, not all experiments affirm these predictions. For example, the A and J series PG both repress NFB transcription and inhibit IB kinase (1, 2); however, only ⌬12-PGJ 2 is anti-inflammatory (11). Likewise, the A and J series PG both repress p53 transcription; however, only the A series PG antagonize p53-dependent apoptosis (Ref. 4 and see below).Herein we report the discovery of a molecular mechanism that clarifies the distinctive cellular effects of cyclopentenone PG. Namely, J series PG preferentially inhibit the ubiquitin isopeptidase activity (ubiquitin-specific protease) of the proteasome pathway. This pathway is the major nonlysosomal degradation pathway in cells (12, 13). The degradation of target proteins via this pathway largely depends on their covalent modification with a ubiquitin polymer. This polymer consists of ubiquitin (8.5 kDa) subunits that are...