Brian Y. Lee

Identification of candidate biomarkers of therapeutic response to docetaxel by proteomic profiling.

Docetaxel chemotherapy improves symptoms and survival in men with metastatic hormone-refractory prostate cancer (HRPC). However, approximately 50% of patients do not respond to Docetaxel and are exposed to significant toxicity without direct benefit. This study aimed to identify novel therapeutic targets and predictive biomarkers of Docetaxel resistance in HRPC. We used iTRAQ-mass spectrometry analysis to identify proteins associated with the development of Docetaxel resistance using Docetaxel-sensitive PC3 cells and Docetaxel-resistant PC3-Rx cells developed by Docetaxel dose escalation. Functional validation experiments were performed using recombinant protein treatment and siRNA knockdown experiments. Serum/plasma levels of the targets in patient samples were measured by ELISA. The IC(50) for Docetaxel in the PC3-Rx cells was 13-fold greater than the parent PC-3 cell line (P = 0.004). Protein profiling identified MIC-1 and AGR2 as respectively up-regulated and down-regulated in Docetaxel-resistant cells. PC-3 cells treated with recombinant MIC-1 also became resistant to Docetaxel (P = 0.03). Conversely, treating PC3-Rx cells with MIC-1 siRNA restored sensitivity to Docetaxel (P = 0.02). Knockdown of AGR2 expression in PC3 cells resulted in Docetaxel resistance (P = 0.007). Furthermore, increased serum/plasma levels of MIC-1 after cycle one of chemotherapy were associated with progression of the cancer (P = 0.006) and shorter survival after treatment (P = 0.002). These results suggest that both AGR2 and MIC-1 play a role in Docetaxel resistance in HRPC. In addition, an increase in serum/plasma MIC-1 level after cycle one of Docetaxel may be an indication to abandon further treatment. Further investigation of MIC-1 as a biomarker and therapeutic target for Docetaxel resistance in HRPC is warranted.

FAK signaling in human cancer as a target for therapeutics

Focal adhesion kinase (FAK) is a key regulator of growth factor receptor- and integrin-mediated signals, governing fundamental processes in normal and cancer cells through its kinase activity and scaffolding function. Increased FAK expression and activity occurs in primary and metastatic cancers of many tissue origins, and is often associated with poor clinical outcome, highlighting FAK as a potential determinant of tumor development and metastasis. Indeed, data from cell culture and animal models of cancer provide strong lines of evidence that FAK promotes malignancy by regulating tumorigenic and metastatic potential through highly-coordinated signaling networks that orchestrate a diverse range of cellular processes, such as cell survival, proliferation, migration, invasion, epithelial-mesenchymal transition, angiogenesis and regulation of cancer stem cell activities. Such an integral role in governing malignant characteristics indicates that FAK represents a potential target for cancer therapeutics. While pharmacologic targeting of FAK scaffold function is still at an early stage of development, a number of small molecule-based FAK tyrosine kinase inhibitors are currently undergoing pre-clinical and clinical testing. In particular, PF-00562271, VS-4718 and VS-6063 show promising clinical activities in patients with selected solid cancers. Clinical testing of rationally designed FAK-targeting agents with implementation of predictive response biomarkers, such as merlin deficiency for VS-4718 in mesothelioma, may help improve clinical outcome for cancer patients. In this article, we have reviewed the current knowledge regarding FAK signaling in human cancer, and recent developments in the generation and clinical application of FAK-targeting pharmacologic agents.

Circulating microRNAs are associated with docetaxel chemotherapy outcome in castration-resistant prostate cancer.

Background:Docetaxel is the first-line chemotherapy for castration-resistant prostate cancer (CRPC). However, response rates are ∼50% and determined quite late in the treatment schedule, thus non-responders are subjected to unnecessary toxicity. The potential of circulating microRNAs as early biomarkers of docetaxel response in CRPC patients was investigated in this study.Methods:Global microRNA profiling was performed on docetaxel-resistant and sensitive cell lines to identify candidate circulating microRNA biomarkers. Custom Taqman Array MicroRNA cards were used to measure the levels of 46 candidate microRNAs in plasma/serum samples, collected before and after docetaxel treatment, from 97 CRPC patients.Results:Fourteen microRNAs were associated with serum prostate-specific antigen (PSA) response or overall survival, according to Mann–Whitney U or log-rank tests. Non-responders to docetaxel and patients with shorter survival generally had high pre-docetaxel levels of miR-200 family members or decreased/unchanged post-docetaxel levels of miR-17 family members. Multivariate Cox regression with bootstrapping validation showed that pre-docetaxel miR-200b levels, post-docetaxel change in miR-20a levels, pre-docetaxel haemoglobin levels and visceral metastasis were independent predictors of overall survival when modelled together.Conclusions:Our study suggests that circulating microRNAs are potential early predictors of docetaxel chemotherapy outcome, and warrant further investigation in clinical trials.

Phosphoproteomic Profiling Identifies Focal Adhesion Kinase as a Mediator of Docetaxel Resistance in Castrate-Resistant Prostate Cancer

Docetaxel remains the standard-of-care for men diagnosed with metastatic castrate-resistant prostate cancer (CRPC). However, only approximately 50% of patients benefit from treatment and all develop docetaxel-resistant disease. Here, we characterize global perturbations in tyrosine kinase signaling associated with docetaxel resistance and thereby develop a potential therapeutic strategy to reverse this phenotype. Using quantitative mass spectrometry–based phosphoproteomics, we identified that metastatic docetaxel-resistant prostate cancer cell lines (DU145-Rx and PC3-Rx) exhibit increased phosphorylation of focal adhesion kinase (FAK) on Y397 and Y576, in comparison with parental controls (DU145 and PC3, respectively). Bioinformatic analyses identified perturbations in pathways regulating focal adhesions and the actin cytoskeleton and in protein–protein interaction networks related to these pathways in docetaxel-resistant cells. Treatment with the FAK tyrosine kinase inhibitor (TKI) PF-00562271 reduced FAK phosphorylation in the resistant cells, but did not affect cell viability or Akt phosphorylation. Docetaxel administration reduced FAK and Akt phosphorylation, whereas cotreatment with PF-00562271 and docetaxel resulted in an additive attenuation of FAK and Akt phosphorylation and overcame the chemoresistant phenotype. The enhanced efficacy of cotreatment was due to increased autophagic cell death, rather than apoptosis. These data strongly support that enhanced FAK activation mediates chemoresistance in CRPC, and identify a potential clinical niche for FAK TKIs, where coadministration with docetaxel may be used in patients with CRPC to overcome chemoresistance. Mol Cancer Ther; 13(1); 190–201. ©2013 AACR.

Cytokine profiling of docetaxel-resistant castration-resistant prostate cancer

Background:Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort.Methods:PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response.Results:PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1β, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002).Conclusions:In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.

ELF5 drives lung metastasis in luminal breast cancer through recruitment of Gr1+ CD11b+ myeloid-derived suppressor cells

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Effect of FAK inhibitor VS-6063 (defactinib) on docetaxel efficacy in prostate cancer

Docetaxel, the standard chemotherapy for metastatic castration‐resistant prostate cancer (CRPC) also enhances the survival of patients with metastatic castration‐sensitive prostate cancer (CSPC) when combined with androgen‐deprivation therapy. Focal Adhesion Kinase (FAK) activation is a mediator of docetaxel resistance in prostate cancer cells. The aim of this study was to investigate the effect of the second generation FAK inhibitor VS‐6063 on docetaxel efficacy in pre‐clinical CRPC and CSPC models.

MicroRNAs as potential therapeutics to enhance chemosensitivity in advanced prostate cancer

Docetaxel and cabazitaxel are taxane chemotherapy treatments for metastatic castration-resistant prostate cancer (CRPC). However, therapeutic resistance remains a major issue. MicroRNAs are short non-coding RNAs that can silence multiple genes, regulating several signalling pathways simultaneously. Therefore, synthetic microRNAs may have therapeutic potential in CRPC by regulating genes involved in taxane response and minimise compensatory mechanisms that cause taxane resistance. To identify microRNAs that can improve the efficacy of taxanes in CRPC, we performed a genome-wide screen of 1280 microRNAs in the CRPC cell lines PC3 and DU145 in combination with docetaxel or cabazitaxel treatment. Mimics of miR-217 and miR-181b-5p enhanced apoptosis significantly in PC3 cells in the presence of these taxanes. These mimics downregulated at least a thousand different transcripts, which were enriched for genes with cell proliferation and focal adhesion functions. Individual knockdown of a selection of 46 genes representing these transcripts resulted in toxic or taxane sensitisation effects, indicating that these genes may be mediating the effects of the microRNA mimics. A range of these genes are expressed in CRPC metastases, suggesting that these microRNA mimics may be functional in CRPC. With further development, these microRNA mimics may have therapeutic potential to improve taxane response in CRPC patients.

Abstract A33: Characterization of signaling pathways associated with docetaxel resistance in prostate cancer

Abstract Docetaxel (DTX) is the only treatment option for patients diagnosed with metastatic castration-refractory prostate cancer (CRPC). However, approximately 50% of patients do not respond to DTX, but are exposed to significant toxicity without direct benefit. This study aims to use mass spectrometry (MS) based techniques to characterize the perturbed intracellular signaling, particularly tyrosine phosphorylation, which contributes to the chemoresistant phenotype in CRPC patients, and thereby identify novel therapeutic targets. Two DTX-resistant prostate cancer cell lines (DU145-Rx & PC3-Rx) were established from their parental DTX-sensitive cell lines, DU145 & PC3, respectively, by a dose-escalation protocol. Phosphotyrosine profiling was performed by immunoaffinity coupled LC-MS/MS in combination with stable isotope labeling with amino acid in cell culture (SILAC). The chemosensitive and resistant cells were cultured in “light” and “heavy” SILAC media, respectively, and their isotopic labels were switched in a replicate experiment. Phosphotyrosine peptides were analysed on the state-of-the-art High Performance Liquid Chromatography MS/MS (Orbitrap Velos ETD). Raw MS spectra were quantified using MaxQuant software. DU145-Rx & PC3-Rx displayed 44 and 35-fold higher IC50 values, respectively, and significantly greater colony forming ability, in the presence of DTX, compared to the parental lines. Phosphotyrosine profiling of these cell lines identified a total of 365 phosphosites in the chemoresistant lines, corresponding to 227 proteins. 3 kinases and 3 cytoskeletal proteins showed a significant increase in tyrosine phosphorylation in both DTX-resistant models. These candidate proteins will be further characterized by gain- and loss-of-function approaches, both in vitro and in xenograft models, to elucidate the intracellular mechanism of chemoresistance, and to design therapeutic strategies to reverse the chemoresistant phenotype in CRPC. Citation Format: Brian Yong Lee, Falko Hochgrafe, Mark Raftery, Lisa G. Horvath, Roger J. Daly. Characterization of signaling pathways associated with docetaxel resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A33.