Androgen receptor nuclear localization correlates with AR-V7 mRNA expression in circulating tumor cells (CTCs) from metastatic castration resistance prostate cancer patients.

Androgen receptor (AR) signaling drives prostate cancer (PC) progression and remains active upon transition to castration resistant prostate cancer (CRPC). Active AR signaling is achieved through the nuclear accumulation of AR following ligand binding and through expression of ligand-independent, constitutively active AR splice variants, such as AR-V7, which is the most commonly expressed variant in metastatic CRPC (mCRPC) patients. Most currently approved PC therapies aim to abrogate AR signaling and activity by inhibiting this ligand-mediated nuclear translocation. In a prospective multi-institutional clinical study, we recently showed that taxane based chemotherapy is also capable of impairing AR nuclear localization (ARNL) in circulating tumor cells (CTCs) from CRPC patients, whereas taxane induced decreases in ARNL were associated with response. Thus, quantitative assessment of ARNL in CTCs can be used to monitor therapeutic response in patients and help guide clinical decisions. Here, we describe the development and implementation of quantitative high throughput (QHT) image analysis algorithms to aid in CTC identification and quantitative assessment of percent ARNL (%ARNL). We applied this algorithm to fifteen CRPC patients at the start of taxane chemotherapy, quantified %ARNL in CTCs, and correlated with expression of AR-V7 mRNA (from CTCs enriched via negative, CD45+ depletion of peripheral blood) and with biochemical (prostate specific antigen; PSA) response to taxane chemotherapy. We found that CTCs from AR-V7 positive patients had higher baseline %ARNL compared to CTCs from AR-V7 negative patients, consistent with the constitutive nuclear localization of AR-V7. In addition, lower %ARNL in CTCs at baseline was associated with biochemical response to taxane chemotherapy. High inter- and intra-patient heterogeneity was also observed. As ARNL is required for active AR signaling, the QHT algorithms described herein can provide prognostic and/or predictive value in future clinical studies.

Transferrin receptor-based circulating tumor cell enrichment provides a snapshot of the molecular landscape of solid tumors and correlates with clinical outcomes.

Circulating tumor cells (CTCs) captured from the bloodstream of patients with solid tumors have the potential to accelerate precision oncology by providing insight into tumor biology, disease progression and response to treatment. However, their potential is hampered by the lack of standardized CTC enrichment platforms across tumor types. EpCAM-based CTC enrichment, the most commonly used platform, is limited by EpCAM downregulation during metastasis and the low EpCAM expression in certain tumor types, including the highly prevalent and lethal NSCLC. In this study we demonstrate that Transferrin Receptor (TfR) is a selective, efficient biomarker for CTC identification and capture in patients with prostate, pancreatic and NSCLC. TfR identifies significantly higher CTC counts than EpCAM, and TfR + -CTC enumeration correlates with disease progression in metastatic prostate and pancreatic cancers, and overall survival and osimetrinib-resistance in non-small cell lung cancer (NSCLC). Profiling of TfR + -CTCs provides a snapshot of the molecular landscape of each respective tumor type and identifies potential mechanisms underlying treatment response to EGFR TKi and immune checkpoint inhibitors in NSCLC. One sentence summary: Transferrin Receptor identifies circulating tumor cells in solid tumors.

Androgen receptor variant shows heterogeneous expression in prostate cancer according to differentiation stage.

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 (NanoStringTM) 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.

Microtubule Engagement with Taxane Is Altered in Taxane-Resistant Gastric Cancer.

PURPOSE: 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. EXPERIMENTAL DESIGN: We performed a post hoc 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. RESULTS: 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 in vitro and in patient samples. CONCLUSIONS: 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.

Taxane resistance in prostate cancer is mediated by decreased drug-target engagement.

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.

Expression of AR-V7 and ARv567es in Circulating Tumor Cells Correlates with Outcomes to Taxane Therapy in Men with Metastatic Prostate Cancer Treated in TAXYNERGY.

PURPOSE: Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. The presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTC) may be associated with taxane treatment outcomes.Experimental Design: A novel digital droplet PCR (ddPCR) assay assessed AR-splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR-splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS). RESULTS: Of the 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive, and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7- versus AR-V7+ (78% vs. 58%; P = 0.23) and for ARv567es- versus ARv567es+ (92% vs. 57%; P = 0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from cycle 1 day 1 to day 8 (n = 24), AR-V7+ patients (n = 16) had a 0.4% decrease versus a 12.9% and 26.7% decrease in AR-V7-/ARv567es- (n = 3) and AR-V7-/ARv567es+ (n = 5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 versus 8.48 months for AR-V7- versus AR-V7+ (HR = 0.38; P = 0.01), and 12.71 versus 7.29 months for ARv567es- versus ARv567es+ (HR = 0.37; P = 0.02). For AR-V7+, AR-V7-/ARv567es+, and AR-V7-/ARv567es- patients, median PFS was 8.48, 11.17, and 16.62 months, respectively (P = 0.0013 for trend). CONCLUSIONS: Although detection of both CTC-specific AR-V7 and ARv567es by ddPCR influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. The absence of both variants was associated with the best response and PFS with taxane treatment.See related commentary by Dehm et al., p. 1696.

Randomized, Noncomparative, Phase II Trial of Early Switch From Docetaxel to Cabazitaxel or Vice Versa, With Integrated Biomarker Analysis, in Men With Chemotherapy-Naïve, Metastatic, Castration-Resistant Prostate Cancer.

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.

A folate receptor-targeting nanoparticle minimizes drug resistance in a human cancer model.

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.

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have ßI-tubulin mutations and altered expression of ßII- and ßIII-tubulin isotypes.

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on ß-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the ßI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a ß-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the ß-tubulin isotype composition of the cells was examined. Increased expression of ßII- and ßIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.

Taxane-induced blockade to nuclear accumulation of the androgen receptor predicts clinical responses in metastatic prostate cancer.

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 ß-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.

The protein farnesyltransferase regulates HDAC6 activity in a microtubule-dependent manner.

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 alpha 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 alpha subunit of FTase (FTalphaKD). Interestingly, the FTalphaKD 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.

HFT-T, a targeting nanoparticle, enhances specific delivery of paclitaxel to folate receptor-positive tumors.

Nonspecific distribution of chemotherapeutic drugs (such as paclitaxel) is a major factor contributing to side effects and poor clinical outcomes in the treatment of human head and neck cancer. To develop novel drug delivery systems with enhanced efficacy and minimized adverse effects, we synthesized a ternary conjugate heparin-folic acid-paclitaxel (HFT), loaded with additional paclitaxel (T). The resulting nanoparticle, HFT-T, is expected to retain the antitumor activity of paclitaxel and specifically target folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of paclitaxel. In vitro experiments found that HFT-T selectively recognizes FR-positive human head and neck cancer cell line KB-3-1, displaying higher cytotoxicity compared to the free form of paclitaxel. In a subcutaneous KB-3-1 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably improved antitumor efficacy of paclitaxel. The average tumor volume in the HFT-T treatment group was 92.9 +/- 78.2 mm(3) vs 1670.3 +/- 286.1 mm(3) in the mice treated with free paclitaxel. Furthermore, paclitaxel tumors showed a resurgence of growth after several weeks of treatment, but this was not observed with HFT-T. This indicates that HFT-T could be more effective in preventing tumors from developing drug resistance. No significant acute in vivo toxicity was observed. These results indicate that specific delivery of paclitaxel with a ternary structured nanoparticle (HFT-T) targeting FR-positive tumor is a promising strategy to enhance chemotherapy efficacy and minimize adverse effects.

Differential regulation of protein phosphatase-1(I) by neurabin.

Neurabin is a brain-specific actin and protein phosphatase-1 (PP-1) binding protein that inhibits the purified catalytic subunit of protein phosphatase-1 (PP-1(C)). However, endogenous PP-1 exists primarily as multimeric complexes of PP-1(C) bound to various regulatory proteins that determine its activity, substrate specificity, subcellular localization and function. The major form of endogenous PP-1 in brain is protein phosphatase-1(I) (PP-1(I)), a Mg(2+)/ATP-dependent form of PP-1 that consists of PP-1(C), the inhibitor-2 regulatory subunit, an activating protein kinase and other unidentified proteins. We have identified four PP-1(I) holoenzyme fractions (PP-1(IA), PP-1(IB), PP-1(IC), and PP-1(ID)) in freshly harvested pig brain separable by poly-L-lysine chromatography. Purified recombinant neurabin (amino acid residues 1-485) inhibited PP-1(IB) (IC(50)=1.1 microM), PP-1(IC) (IC(50)=0.1 microM), and PP-1(ID) (IC(50)=0.2 microM), but activated PP-1(IA) by up to threefold (EC(50)=40 nM). The PP-1(IA) activation domain was localized to neurabin(1-210). Our results indicate a novel mechanism of PP-1 regulation by neurabin as both an inhibitor and an activator of distinct forms of PP-1(I) in brain.