Ada Gjyrezi

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 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.

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.

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 βI-tubulin mutations and altered expression of βII- and βIII-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 β-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 βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-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 β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-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. Mol Cancer Ther; 10(8); 1419–29. ©2011 AACR.

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 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.

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.

Abstract 115: An integrated framework of transcriptome analysis leads to mechanistic insights into the FTI/Taxol synergy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC A novel class of anticancer agents, the farnesyltransferase inhibitors (FTIs), has exhibited modest activity in the clinic as single agents, but was found to have promising activity in combination with the microtubule-stabilizing drug Taxol (TX), both in vitro and in vivo models. Recent phase I and II oncology clinical trials with an FTI/TX combination have demonstrated a clinical benefit for a subset of patients refractory to previous taxane-treatment. However, the precise cellular machinery underlying the FTI/TX synergistic drug combination still needs to be better understood. To this end, we undertook an unbiased experimental/computational approach that combined (i) measurements of drug-mediated changes on tubulin biomarkers and overall survival; (ii) whole-genome transcriptome analysis; (iii) utilization of interaction and functional databases to identify functional and pathway connections, (iv) computational modeling and prediction based on an in silico model predictive of drug synergy. Drug-induced transcriptome changes were examined using whole-genome Affymetrix HGU133 Plus 2 microarrays. The raw data were analyzed using two global statistical analysis methods (ANOVA and SAM). Gene changes that passed both tests, using a fold change criterion, were deemed significant and subjected to experimental validation by qPCR and siRNA. The transcriptome changes that were unique to the LNF/TX combination were evaluated in the context of detailed molecular interaction maps and groups of biological function. Our analyses identified several major network hubs that include: (i) repression of the mitotic spindle-checkpoint kinase, polo-like kinase 1 (Plk1) which is also implicated in carcinogenesis and was shown to directly bind and phosphorylate α- and β-tubulins. (ii) overexpression of CDKN1A and MDM2, both of which are shown to be inversely regulated by Plk1; (iii) overexpression of the class II beta tubulin isotype TUBB2A and of several heat shock protein such as HSPA1A and DNAJB9 and (iv) induction of the focal adhesion molecule paxillin (PXN). Interestingly, stable knockdown of protein farnesyltransferase mimicked the effects of FTI treatment and resulted in strong substratum attachment and migration defects suggesting that paxillin could be regulated by farnesyltransferase and its pharmacologic inhibitors. Functional studies to elucidate this mechanism are currently underway. In summary, we demonstrate a combined experimental and computational approach that includes functional analysis, data-driven simulation and experimental validation, and its power to identify key cellular components, both known and novel, in the anticancer activity of drug combinations exhibiting therapeutic synergy. (Supported by NCI P50 CA128613 & P01 CA116676) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 115.

Abstract 2736: A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients

Prostate cancer (PC) is the second leading cause of cancer death in men in the US. The aberrant functioning of androgen receptor signaling is the central driving force behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. Hence, androgen deprivation therapy (ADT) is the first line of treatment for PC patients. However, many patients progress becoming resistant to ADT therapy, due to the expression of AR splice variants (AR-Vs), which lack the ligand binding domain and are constitutively active in the nucleus. Expression of the AR splice variant, AR-v7, in circulating tumor cells (CTCs) isolated from the blood of PC patients was correlated with resistance to enzalutamide and abiraterone, which are the next generation AR signaling inhibitors in CRPC. Further, there is evidence that AR-Vs may convey cross-resistance, not only to enzalutamide and abiraterone, but also to taxanes, highlighting that their assessment in the clinic may have clinical utility. We developed a novel, specific and highly sensitive assay to measure mRNA expression of the AR full length (AR-FL) and the splice variants ARv7 and ARv567es, by using Droplet Digital PCR in CTCs isolated from CRPC patients. The analytical specificity of the assay was determined by transfecting cells with plasmids encoding AR-FL, AR-v7 and AR-v567 and showed that each probe detected signal only in cells expressing the respective transcript. No signal was detected against genomic DNA, indicating lack of non-specific binding. Also, the assay detected endogenous expression of AR-FL and AR-v7 in VCAP or 22RV1 cells, while no variant expression was detected in healthy donor blood. The analytical sensitivity of the assay was determined in a series of serial dilution experiments that showed sensitivity down to single cell. We then used this assay to determine the clinical prevalence and expression pattern of each of these variants in CTCs from about 200 mCRPC patient samples and blood from 40 healthy donors. CTCs were enriched by EpCAM- or PSMA-based positive selection or CD45 negative depletion in an antigen-agnostic manner. AR-FL was detected in ~80% of mCRPC samples irrespective of CTC-enrichment technology. AR-v7 was expressed in 65% of the samples in which in CTCs were enriched either by PSMA-positive selection or by negative depletion. In contrast, EpCAM-based CTC enrichment showed lower AR-v7 expression both in terms of expression levels and prevalence. In addition, CTC enrichment following negative depletion showed that 30% of the samples had higher AR-v7 expression levels as compared to AR-FL. This expression pattern was not observed in the samples using EpCAM-based selection. Collectively, these data suggest distinct CTC subpopulations are present in CRPC patient samples, with differential expression of AR-Vs that could have important predictive and prognostic implications. Citation Format: Ada Gjyrezi, Giuseppe Galletti, Areti Strati, Seaho Kim, Evi Lianidou, David M. Nanus, Jun Luo, Emmanuel Antonarakis, Scott T. Tagawa, Andrew Armstrong, Paraskevi Giannakakou. A digital droplet PCR assay for the quantitation of androgen receptor and splice variant expression in CTCs from metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2736. doi:10.1158/1538-7445.AM2017-2736

Abstract 3489: Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells

Prostate cancer is the most commonly diagnosed male cancer in the United States. Taxanes are the only established chemotherapy drugs proven to be effective in improving survival of men with advanced prostate cancer through disruption of the AR-signaling axis downstream of microtubule stabilization. However, there is significant heterogeneity in how patients respond to taxanes and most patients ultimately become refractory due to the development of drug resistance. Currently, the molecular basis of clinical taxane resistance in PC is poorly understood. Prostate cancer circulating tumor cells (P-CTCs) are often found in the peripheral blood of patients suffering from metastatic prostate cancer and have been clinically used as prognostic biomarker for metastatic progression and treatment outcome. The objective of this study is to identify clinically relevant mechanisms of taxane resistance through conducting RNA-Seq analysis in P-CTCs isolated from patients before, during and after they become refractory to taxane chemotherapy. To show feasibility of RNA-Seq experiments with limiting samples such as CTCs and given the presence of contaminating leucocytes, a pilot experiment was performed in which limiting numbers of prostate cancer cells (LNCaP) either pure or enriched following spiking into healthy donor blood, were analyzed by RNA-Seq. Matching healthy donor blood processed with the same enrichment protocol was used as germline control as well as control for the presence of contaminating leucocytes following CTC enrichment. Trimmed reads were aligned to human reference genome (hg38) using STAR. Determination of Fragments Per Kilobase of exon per Million mapped fragments (FPKM) was performed using Cufflinks and heat map was built based on the value of log10(FPKM+1). Gene expression analysis showed that markers of prostate (such as AR, PSMA, KLK3, KLK2, and AMACR) or epithelial lineage (such as EpCAM, CDH1, KRT8 and KRT18) were detected in both pure LNCaP cells-regardless of amount- as well as limited number of captured LNCaP cells in the presence of contaminating leucocytes. In contrast, healthy donor blood was negative for the prostate and epithelial lineage markers and positive for the leucocyte specific markers (such as CD45 and CD16). Gene set enrichment analysis (GSEA) indicated significant enrichment for Andorgen response, MYC, MTOR and RB related pathways in pure and captured LNCap cells compared with healthy donor blood. These data clearly show that by using RNA-Seq we can detect the prostate and epithelial specific gene signatures of limited number of spiked prostate cancer cells using the microfluidic device. Ongoing work includes RNA-Seq analysis of P-CTCs isolated from patients before and after taxane treatment, in order to detect differentially expressed genes, pathways, and potentially driver somatic mutations associated with clinical taxane resistance. Citation Format: Jiaren Zhang, Ada Gjyrezi, Prashant Thakkar, Giuseppe Galletti, Akanksha Verma, Olivier Elemento, Paraskevi Giannakakou. Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3489.

Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C 14 -labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Diaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600