Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel + androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-κB transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44⁺ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44⁺ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evidence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer. Mol Cancer Ther; 13(5); 1270–84. ©2014 AACR.

The complete structures of LPS core types 1 and 2 fromKlebsiella pneumoniaehave been described by other authors. They are characterized by a lack of phosphoryl residues, but they contain galacturonic acid (GalA) residues, which contribute to the necessary negative charges. The presence of a capsule was determined in core-LPS non-polar mutants from strains 52145 (O1 : K2), DL1 (O1 : K1) and C3 (O8 : K66). O-antigen ligase (waaL) mutants produced a capsule. Core mutants containing the GalA residues were capsulated, while those lacking the residues were non capsulated. Since the proteins involved in the transfer of GalA (WabG) and glucosamine residues (WabH) are known, the chemical basis of the capsular-K2–cell-surface association was studied. Phenol/water extracts fromK. pneumoniae52145ΔwabH waaLand 52145ΔwaaLmutants, but not those from fromK. pneumoniae52145ΔwabG waaLmutant, contained both LPS and capsular polysaccharide, even after hydrophobic chromatography. The two polysaccharides were dissociated by gel-filtration chromatography, eluting with detergent and metal-ion chelators. From these results, it is concluded that the K2 capsular polysaccharide is associated by an ionic interaction to the LPS through the negative charge provided by the carboxyl groups of the GalA residues.

The Aeromonas hydrophila type III secretion system (T3SS) has been shown to play a crucial role in this pathogen's interactions with its host. We previously described the genetic organization of the T3SS cluster and the existence of at least one effector, called AexT, in A. hydrophila strain AH-3. In this study, we analyzed the expression of the T3SS regulon by analyzing the activity of the aopN-aopD and aexT promoters (T3SS machinery components and effector, respectively) by means of two different techniques: promoterless gfp fusions and real-time PCR. The expression of the A. hydrophila AH-3 T3SS regulon was induced in response to several environmental factors, of which calcium depletion, a high magnesium concentration, and a high growth temperature were shown to be the major ones. Once the optimal conditions were established, we tested the expression of the T3SS regulon in the background of several virulence determinant knockouts of strain AH-3. The analysis of the data obtained from axsA and aopN mutants, both of which have been described to be T3SS regulators in other species, allowed us to corroborate their function as the major transcription regulator and valve of the T3SS, respectively, in Aeromonas hydrophila . We also demonstrated the existence of a complicated interconnection between the expression of the T3SS and several other different virulence factors, such as the lipopolysaccharide, the PhoPQ two-component system, the ahyIR quorum sensing system, and the enzymatic complex pyruvate deshydrogenase. To our knowledge, this is the first study of the A. hydrophila T3SS regulatory network.

Taxanes are the most active chemotherapy agents in metastatic castration-resistant prostate cancer (mCRPC) patients; yet, resistance occurs almost invariably, representing an important clinical challenge. Taxane-platinum combinations have shown clinical benefit in a subset of patients, but the mechanistic basis and biomarkers remain elusive. To identify mechanisms and response indicators for the antitumor efficacy of taxane-platinum combinations in mCRPC. Transcriptomic data from a publicly available mCRPC dataset of taxane-exposed and taxane-naïve patients were analyzed to identify response indicators and emerging vulnerabilities. Functional and preclinical validation was performed in taxane-resistant mCRPC cell lines and genetically engineered mouse models (GEMMs). Metastatic CRPC cells were treated with docetaxel, cisplatin, carboplatin, the CXCR2 antagonist SB265610, and the BCL-2 inhibitor venetoclax. Gain and loss of function in culture of CXCR2 and BCL-2 were achieved by overexpression or siRNA silencing. Preclinical assays in GEMM mice tested the antitumor efficacy of taxane-platinum combinations. Proliferation, apoptosis, and colony assays measured drug activity in vitro. Preclinical endpoints in mice included growth, survival, and histopathology. Changes in CXCR2, BCL-2, and chemokines were analyzed by reverse transcriptase quantitative polymerase chain reaction and Western blot. Human expression data were analyzed using Gene Set Enrichment Analysis, hierarchical clustering, and correlation studies. GraphPad Prism software and R-studio were used for statistical and data analyses. Transcriptomic data from taxane-exposed human mCRPC tumors correlate with a marked negative enrichment of apoptosis and inflammatory response pathways accompanied by a marked downregulation of CXCR2 and BCL-2. Mechanistically, we show that docetaxel inhibits CXCR2 and that BCL-2 downregulation occurs as a downstream effect. Further, we demonstrated in experimental models that the sensitivity to cisplatin is dependent on CXCR2 and BCL-2, and that targeting them sensitizes prostate cancer (PC) cells to cisplatin. In vivo taxane-platinum combinations are highly synergistic, and previous exposure to taxanes sensitizes mCRPC tumors to second-line cisplatin treatment. The hitherto unappreciated attenuation of the CXCR2/BCL-2 axis in taxane-treated mCRPC patients is an acquired vulnerability with potential predictive activity for platinum-based treatments. A subset of patients with aggressive and therapy-resistant prostate cancer benefits from taxane-platinum combination chemotherapy; however, we lack the mechanistic understanding of how that synergistic effect occurs. Here, using patient data and preclinical models, we found that taxanes reduce cancer cell escape mechanisms to chemotherapy-induced cell death, hence making these cells more vulnerable to additional platinum treatment.

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae contains two galacturonic acid (GalA) residues, but only one GalA transferase (WabG) has been identified. Data from chemical and structural analysis of LPS isolated from a wabO mutant show the absence of the inner core β-GalA residue linked to l - glycero - d - manno -heptose III ( l , d -Hep III). An in vitro assay demonstrates that the purified WabO is able to catalyze the transfer of GalA from UDP-GalA to the acceptor LPS isolated from the wabO mutant, but not to LPS isolated from waaQ mutant (deficient in l , d -Hep III). The absence of this inner core β-GalA residue results in a decrease in virulence in a capsule-dependent experimental mouse pneumonia model. In addition, this mutation leads to a strong reduction in cell-bound capsule. Interestingly, a K66 Klebsiella strain (natural isolate) without a functional wabO gene shows reduced levels of cell-bound capsule in comparison to those of other K66 strains. Thus, the WabO enzyme plays an important role in core LPS biosynthesis and determines the level of cell-bound capsule in Klebsiella pneumoniae .