Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival ...in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired β-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy.
Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for ...treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform (NanoString
) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.
The cytoplasmic deacetylase HDAC6 is an important regulator of cellular pathways that include response to stress, protein folding, microtubule stability, and cell migration, thus representing an ...attractive target for cancer chemotherapy. However, little is known about its upstream regulation. Our previous work has implicated HDAC6 as a new protein target for the farnesyltransferase inhibitors (FTIs), although HDAC6 lacks a farnesylation motif. Here we show that the protein farnesyltransferase (FTase) and HDAC6 are present in a protein complex together with microtubules in vivo and in vitro. FTase binds microtubules directly via its α subunit, and this association requires the C terminus of tubulin. Treatment with an FTI removed FTase, but not HDAC6, from the protein complex, suggesting that the active form of FTase is bound to microtubules. Importantly, the removal of FTase from microtubules abrogated HDAC6 activity, as did a stable knockdown of the α subunit of FTase (FTαKD). Interestingly, the FTαKD cells showed increased sensitivity to the antiproliferative effects of Taxol and the FTI lonafarnib when used either as single agents or in combination as compared with parental cells. Altogether, these data suggest that FTase, via its tubulin-association, is a critical upstream regulator of HDAC6 activity and that FTase expression could help stratify cancer patients that would most benefit from this treatment.
Although taxane-based therapy is standard treatment for advanced gastric cancer, a majority of patients exhibit intrinsic resistance to taxanes. Here, we aim to identify the molecular basis of taxane ...resistance in gastric cancer.
We performed a
analysis of the TAX-325 clinical trial and molecular interrogation of gastric cancer cell lines to assess the benefit of docetaxel in diffuse (DIF-GC) versus intestinal (INT-GC) gastric cancer. We assessed drug-induced microtubule stabilization in gastric cancer cells and in biopsies of patients with gastric cancer treated with taxanes. We performed transcriptome analysis in taxane-treated gastric cancer cells and patients to identify molecular drivers of taxane resistance.
Patients with DIF-GC did not derive a clinical benefit from taxane treatment suggesting intrinsic taxane resistance. DIF-GC cell lines displayed intrinsic resistance specific to taxanes because of impaired drug-induced microtubule stabilization, in the absence of tubulin mutations or decreased drug accumulation. Using taxane-treated gastric cancer patient biopsies, we demonstrated that absence of drug-target engagement was correlated with clinical taxane resistance. Taxane-sensitive cell lines displayed faster microtubule dynamics at baseline, implicating proteins that regulate cytoskeletal dynamics in intrinsic taxane resistance. Differential gene expression analysis of untreated and docetaxel-treated gastric cancer lines and patient samples identified kinesins to be associated with taxane sensitivity
and in patient samples.
Our data reveal that taxane resistance is more prevalent in patients with DIF-GC, support assessment of drug-target engagement as an early read-out of taxane clinical efficacy, and encourage the investigation of kinesins and other microtubule-associated proteins as potentially targetable mediators of taxane resistance in gastric cancer.
Despite widespread use of taxanes, mechanisms of action and resistance in vivo remain to be established, and there is no way of predicting who will respond to therapy. This study examined prostate ...cancer (PCa) xenografts and patient samples to identify in vivo mechanisms of taxane action and resistance. Docetaxel drug-target engagement was assessed by confocal anti-tubulin immunofluorescence to quantify microtubule bundling in interphase cells and aberrant mitoses. Tumor biopsies from metastatic PCa patients obtained 2 to 5 days after their first dose of docetaxel or cabazitaxel were processed to assess microtubule bundling, which correlated with clinical response. Microtubule bundling was evident in PCa xenografts 2 to 3 days after docetaxel treatment but was decreased or lost with acquired resistance. Biopsies after treatment with leuprolide plus docetaxel showed extensive microtubule bundling as did biopsies obtained 2 to 3 days after initiation of docetaxel or cabazitaxel in 2 patients with castration-resistant PCa with clinical responses. In contrast, microtubule bundling in biopsies 2 to 3 days after the first dose of docetaxel was markedly lower in 4 nonresponding patients. These findings indicate that taxanes target both mitotic and interphase cells in vivo and that resistance is through mechanisms that impair drug-target engagement. Moreover, the findings suggest that microtubule bundling after initial taxane treatment may be a predictive biomarker for clinical response.
Resistance to chemotherapy is a major obstacle in cancer therapy. The main purpose of this study is to evaluate the potential of a folate receptor-targeting nanoparticle to overcome/minimize drug ...resistance and to explore the underlying mechanisms. This is accomplished with enhanced cellular accumulation and retention of paclitaxel (one of the most effective anticancer drugs in use today and a well-known P-glycoprotein (P-gp) substrate) in a P-gp-overexpressing cancer model. The folate receptor-targeted nanoparticle, HFT-T, consists of a heparin-folate-paclitaxel (HFT) backbone with an additional paclitaxel (T) loaded in its hydrophobic core. In vitro analyses demonstrated that the HFT-T nanoparticle was superior to free paclitaxel or nontargeted nanoparticle (HT-T) in inhibiting proliferation of P-gp-overexpressing cancer cells (KB-8-5), partially due to its enhanced uptake and prolonged intracellular retention. In a subcutaneous KB-8-5 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably prolonged retention within tumor tissues. Importantly, HFT-T treatment markedly retarded tumor growth in a xenograft model of resistant human squamous cancer. Immunohistochemical analysis further indicated that increased in vivo efficacy of HFT-T nanoparticles was associated with a higher degree of microtubule stabilization, mitotic arrest, antiangiogenic activity, and inhibition of cell proliferation. These findings suggest that when the paclitaxel was delivered as an HFT-T nanoparticle, the drug is better retained within the P-gp-overexpressing cells than the free form of paclitaxel. These results indicated that the targeted HFT-T nanoparticle may be promising in minimizing P-gp related drug resistance and enhancing therapeutic efficacy compared with the free form of paclitaxel.
Abstract
Androgen receptor (AR) drives prostate cancer (PC) progression, even in castrate-conditions due to expression of AR variants (AR-V), which are constitutively active in the nucleus. ...Castration-resistant prostate cancer (CRPC) is currently lethal, due to the development of drug resistance. The molecular basis of clinical drug resistance remains poorly elucidated, due to tumor heterogeneity and limited availability of serial tumor sampling as disease progresses. Circulating tumor cells (CTCs), isolated from patient peripheral blood, have emerged as a reliable, easy to obtain source of tumor cells which can be and used to interrogate the molecular disease evolution. To date, EpCAM-based CTC enrichment is commonly used but has important shortcomings as EpCAM is often lost during EMT, thus, missing the more aggressive mesenchymal CTCs. On the other hand, Transferrin Receptor (TfR) which is overexpressed in cancer cells, is a cell surface protein required for iron uptake and is critical for several biological processes such as cell proliferation and metabolism. Mining large gene expression databases in PC we observed that TfR expression increased with disease stage, from adenocarcinoma (TCGA) to CRPC (SU2C) to the more aggressive neuroendocrine PC (NEPC), while EpCAM expression remained unchanged. We also found that TfR expression was increased in EMT cell models, while EpCAM expression was lost, suggesting that TfR may perform well in CTC isolation across the EMT spectrum. We then tested TfR- and EpCAM-based CTC enrichment in a cohort of 31 CRPC patients, where we observed a significantly higher number of TfR+ CTCs (median, 304) than EpCAM+-CTCs (median, 21) (p<0.01). RNA-sequencing of TfR+ CTCs in comparison to EpCAM+ CTCs, followed by gene-set enrichment analyses revealed that the TfR+ subpopulation was significantly enriched in prostate specific oncogenic pathways including EMT, suggesting distinct molecular profiles between the two subsets.
To investigate the clinical relevance of the two CTC subsets, we quantitated AR-FL and splice variants AR-V7 and AR-v567es expression by digital droplet PCR (ddPCR) in pools of TfR+-CTCs vs EpCAM+-CTCs. Our results from 16 CRPC patients showed that while expression of AR-FL was similar in the two CTC subsets, both AR variants were enriched in TfR+-CTCs. To better map the tumor heterogeneity of AR-V expression we isolated 102 single TfR+- and 63 single EpCAM+-CTCs from 3 CRPC patients and quantified AR-V expression by ddPCR. The single CTC analysis revealed that AR-V7 and AR-v567es were expressed in 21% and 18% of TfR+-CTCs, respectively, vs 0% in EpCAM+-CTCs. These data identify TfR as a promising marker for CTC identification across the EMT gradient, and provide a unique insight into tumor cell heterogeneity, which was not appreciated before, and which may have significant clinical impact.
A. Gjyrezi and G. Galletti contributed equally to this work.
Citation Format: Ada Gjyrezi, Giuseppe Galletti, Jiaren Zhang, Rohan Bareja, Ahmed Halima, Daniel Worroll, David Nanus, Scott Tagawa, Himisha Beltran, Paraskevi Giannakakou. Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 451.
Purpose The TAXYNERGY trial ( ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of ...sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve ≥ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed ≥ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. Results Sixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor-targeted therapy. Overall, 35 patients (55.6%) had confirmed ≥ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had ≥ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve ≥ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved ≥ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of ≥ 50% PSA decrease at C4 ( P = .009). Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). Conclusion The early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.
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