Luigi Portella

Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer

Prostate cancer growth depends on androgen receptor signaling. Androgen ablation therapy induces expression of constitutively active androgen receptor splice variants that drive disease progression. Taxanes are a standard of care therapy in castration-resistant prostate cancer (CRPC); however, mechanisms underlying the clinical activity of taxanes are poorly understood. Recent work suggests that the microtubule network of prostate cells is critical for androgen receptor nuclear translocation and activity. In this study, we used a set of androgen receptor deletion mutants to identify the microtubule-binding domain of the androgen receptor, which encompasses the DNA binding domain plus hinge region. We report that two clinically relevant androgen receptor splice variants, ARv567 and ARv7, differentially associate with microtubules and dynein motor protein, thereby resulting in differential taxane sensitivity in vitro and in vivo. ARv7, which lacks the hinge region, did not co-sediment with microtubules or coprecipitate with dynein motor protein, unlike ARv567. Mechanistic investigations revealed that the nuclear accumulation and transcriptional activity of ARv7 was unaffected by taxane treatment. In contrast, the microtubule-interacting splice variant ARv567 was sensitive to taxane-induced microtubule stabilization. In ARv567-expressing LuCap86.2 tumor xenografts, docetaxel treatment was highly efficacious, whereas ARv7-expressing LuCap23.1 tumor xenografts displayed docetaxel resistance. Our results suggest that androgen receptor variants that accumulate in CRPC cells utilize distinct pathways of nuclear import that affect the antitumor efficacy of taxanes, suggesting a mechanistic rationale to customize treatments for patients with CRPC, which might improve outcomes.

Preclinical development of a novel class of CXCR4 antagonist impairing solid tumors growth and metastases.

The CXCR4/CXCL12 axis plays a role in cancer metastases, stem cell mobilization and chemosensitization. Proof of concept for efficient CXCR4 inhibition has been demonstrated in stem cell mobilization prior to autologous transplantation in hematological malignancies. Nevertheless CXCR4 inhibitors suitable for prolonged use as required for anticancer therapy are not available. To develop new CXCR4 antagonists a rational, ligand-based approach was taken, distinct from the more commonly used development strategy. A three amino acid motif (Ar-Ar-X) in CXCL12, also found in the reverse orientation (X-Ar-Ar) in the vMIP-II inhibitory chemokine formed the core of nineteen cyclic peptides evaluated for inhibition of CXCR4-dependent migration, binding, P-ERK1/2-induction and calcium efflux. Peptides R, S and I were chosen for evaluation in in vivo models of lung metastases (B16-CXCR4 and KTM2 murine osteosarcoma cells) and growth of a renal cells xenograft. Peptides R, S, and T significantly reduced the association of the 12G5-CXCR4 antibody to the receptor and inhibited CXCL12-induced calcium efflux. The four peptides efficiently inhibited CXCL12-dependent migration at concentrations as low as 10 nM and delayed CXCL12-mediated wound healing in PES43 human melanoma cells. Intraperitoneal treatment with peptides R, I or S drastically reduced the number of B16-CXCR4-derived lung metastases in C57/BL mice. KTM2 osteosarcoma lung metastases were also reduced in Balb/C mice following CXCR4 inhibition. All three peptides significantly inhibited subcutaneous growth of SN12C-EGFP renal cancer cells. A novel class of CXCR4 inhibitory peptides was discovered. Three peptides, R, I and S inhibited lung metastases and primary tumor growth and will be evaluated as anticancer agents.

Regulatory T cells, interleukin (IL)‐6, IL‐8, Vascular endothelial growth factor (VEGF), CXCL10, CXCL11, epidermal growth factor (EGF) and hepatocyte growth factor (HGF) as surrogate markers of host immunity in patients with renal cell carcinoma

To identify a phenotype that could be informative and prognostic in patients with renal cell carcinoma (RCC) peripheral blood was evaluated for TH1, TH2, regulatory T cells (Tregs), natural killer (NK) and NKT cells and for cytokines/chemokines.

Circulating Tumor Cells in Prostate Cancer Diagnosis and Monitoring: An Appraisal of Clinical Potential

Circulating tumor cells (CTCs) have emerged as a viable solution to the lack of tumor tissue availability for patients with a variety of solid tumors, including prostate cancer. Different approaches have been used to capture this tumor cell population and several of these techniques have been used to assess the potential role of CTCs as a biological marker to predict treatment efficacy and clinical outcome. CTCs are now considered a strong tool to understand the molecular characteristics of prostate cancer, and to be used and analyzed as a ‘liquid biopsy’ in the attempt to grasp the biological portrait of the disease in the individual patient.

CXCR4 expression affects overall survival of HCC patients whereas CXCR7 expression does not

Hepatocellular carcinoma (HCC) is a heterogeneous disease with a poor prognosis and limited markers for predicting patient survival. Because chemokines and chemokine receptors play numerous and integral roles in HCC disease progression, the CXCR4–CXCL12–CXCR7 axis was studied in HCC patients. CXCR4 and CXCR7 expression was analyzed by immunohistochemistry in 86 HCC patients (training cohort) and validated in 42 unrelated HCC patients (validation cohort). CXCR4 levels were low in 22.1% of patients, intermediate in 30.2%, and high in 47.7%, whereas CXCR7 levels were low in 9.3% of patients, intermediate in 44.2% and high in 46.5% of the patients in the training cohort. When correlated to patient outcome, only CXCR4 affected overall survival (P=0.03). CXCR4–CXCL12–CXCR7 mRNA levels were examined in 33/86 patients. Interestingly, the common CXCR4–CXCR7 ligand CXCL12 was expressed at significantly lower levels in tumor tissues compared to adjacent normal liver (P=0.032). The expression and function of CXCR4 and CXCR7 was also analyzed in several human HCC cell lines. CXCR4 was expressed in Huh7, Hep3B, SNU398, SNU449 and SNU475 cells, whereas CXCR7 was expressed in HepG2, Huh7, SNU449 and SNU475 cells. Huh7, SNU449 and SNU475 cells migrated toward CXCL12, and this migration was inhibited by AMD3100/anti-CXCR4 and by CCX771/anti-CXCR7. Moreover, SNU449 and Huh7 cells exhibited matrix invasion in the presence of CXCL12 and CXCL11, a ligand exclusive to CXCR7. In conclusion, CXCR4 affects the prognosis of HCC patients but CXCR7 does not. Therefore, the CXCR4–CXCL12–CXCR7 axis plays a role in the interaction of HCC with the surrounding normal tissue and represents a suitable therapeutic target.Cellular & Molecular Immunology advance online publication, 3 November 2014; doi:10.1038/cmi.2014.102

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 novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

Abstract 580: Nivolumab treatment of metastatic renal cancer patients impairs Tregs and potentiates NK function: The role of CXCR4 inhibition (“REVOLUTION Trial”)

Despite encouraging results, Nivolumab response is not as wide as expected in renal cancer ( RCC). Among mechanisms of immunoresistance T-regulatory cells(Tregs) activity plays a central role. We previously showed that tumoral Tregs in RCC patients are more suppressive than healthy donors Tregs. Since tumoral Tregs express high level of CXCR4, the receptor antagonism was evaluated on patients Tregs revealing that CXCR4 antagonism reverted Treg suppressive activity( Santagata et al, submitted manuscript). To identify biomarkers informative and predictive of nivolumab efficacy Treg function, tumoral access and NK interactions was determined in nivolumab treated mRCC patients (“ Revolution” trial). At today eleven patients were analyzed for Tregs and NKs function at time 0-2-4 weeks-3-6 months of treatment. 2 patients died for unrelated causes, 5 Patients showed PR, 4 SD and 1 PD at six months. At time 0 Tregs function, evaluated as inhibition of T-effector (Teff) proliferation, varied among patients; two patients showed anergy reversed by Nivolumab treatment. Overall, increase in Teff proliferation, compatible with a decreased Tregs activity, was revealed during nivolumab treatment and, while the total peripheral Tregs was unaffected, a decrease in CD4+CD25+127lowFOXp3 highCD45RA - (suppressive Tregs) was detected. NK activity, evaluated as CD107a exposure, increased with nivolumab treatment with a concomitant reduction in the inhibitory receptors CD158a, PD-1 and CXCR4. Moreover patients Tregs and NKs were evaluated in vitro for the efficacy of CXCR4 inhibition in affecting cell function. CXCR4 antagonists suppressed the Treg mediated inhibition of Teff proliferation. In conclusion the evaluation of 11 patients enrolled in the REVOLUTION study revealed detectable variations on Tregs and NKs function relatively to Nivolumab treatment to be correlated with clinical outcome. Tregs suppressive function was impaired by inhibition of CXCR4 receptor. Citation Format: Sara Santagata, Anna Maria Trotta, Maria Napolitano, Luigi Portella, Sabrina Rossetti, Sisto Perdona, Sandro Pignata, Stefania Scala. Nivolumab treatment of metastatic renal cancer patients impairs Tregs and potentiates NK function: The role of CXCR4 inhibition (“REVOLUTION Trial”) [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 580. doi:10.1158/1538-7445.AM2017-580

Abstract 3486: Dissecting the mechanism of AR-v7 nuclear translocation in prostate cancer

Continuous androgen receptor (AR) signaling is the key driver of castration-resistant prostate cancer (CRPC), despite prior androgen deprivation therapy (ADT). Potent, next-generation AR signaling inhibitors, such as abiraterone and enzalutamide have been recently added to the standard of care in the treatment of prostate cancer. However, resistance to these drugs inevitably emerges and is mediated by adaptive mechanisms that restore AR function, in the form of constitutively active, ligand-independent AR-variant (AR-V) overexpression. The microtubule-targeting drugs Docetaxel (DTX) and Cabazitaxel (CTX) are the only chemotherapy that significantly improves survival of CRPC patients. We have shown that full-length AR (AR-fl) binds microtubules (MTs) via its hinge domain, and utilizes them as tracks to facilitate its nuclear translocation. Taxanes keep AR-fl inactive in the cytoplasm as a result of MT stabilization. Of the two most prevalent AR-Vs, ARv567 contains the hinge region and is sensitive to taxane treatment while AR-v7, expressed in 60% of CRPC patients, lacks the hinge region and does not depend on MTs for its nuclear translocation. Therefore, ARv7 nuclear translocation is MT-independent and neither taxane treatment nor the disruption of dynein motor complex inhibited its nuclear localization. Thus, AR-v7 expression confers taxane resistance in vivo, in addition to next-generation AR inhibitors. Hence, inhibition of AR-v7 nuclear translocation and activity is critical to overcome drug resistance to current therapeutic modalities. However, the mechanism of ARv7 nuclear translocation is not clearly understood. Previously, we showed that ARv7 exhibited faster kinetics of nuclear translocation compared to AR-fl. Additionally, AR-v7 nuclear import is independent of the importin-α/β pathway, which is utilized by the MT-dependent, AR-fl and ARv567. Upon disruption of the Ran-GTPase nuclear import pathway, by overexpression of the catalytic mutant Q69L form of Ran, we observed that the nuclear import of both AR-fl and AR-V567 was significantly impaired, while ARv7 nuclear import was only partially affected. In this study, we hypothesized that molecular dynamics of AR nuclear import and export determines overall spatiotemporal localization of AR and its activity. To test this hypothesis, we generated photo-convertible AR-fl and AR-Vs (AR-v567 and AR-v7) fused to mEos4b construct, which enables us to track protein mobilization in the region of interest after photo-conversion in live cells. We are currently studying the nuclear import and export kinetics of both AR-fl and AR variants (AR-v567 and AR-v7) alone and in combination using the photo-conversion methodology. Elucidation of the distinct pathway(s) that mediate ARv7 nuclear import and/or export will allow the rationale design of effective ARv7 inhibitors or the development of co-targeting strategies in combination with taxanes and AR signaling inhibitors. Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Luigi Portella, Paraskevi Giannakakou. Dissecting the mechanism of AR-v7 nuclear translocation in prostate cancer. [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 3486.

Abstract 3451: Distinct nuclear translocation mechanism of androgen receptor variant ARv7

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Next generation androgen receptor (AR) inhibitors (abiraterone, enzalutamide) are used to treat metastatic prostate cancer (mCRPC) patients with a response rate of 20∼40%. The presence of androgen receptor variants has emerged as one of the mechanisms of resistance to these drugs. Specifically, the ARv7 and Arv567 splice variants lacking the ligand-binding domain are constitutively active in the nucleus, and have been identified in mCRPC patients. Recently, ARv7 expression in circulating tumor cells (CTCs) from mCRPC patients was correlated with resistance to abiraterone and enzalutamide. Following androgen deprivation therapy (ADT), CRPC patients receive taxane chemotherapy as first and second line of treatment. We have recently shown that AR utilizes microtubules and the dynein motor protein as transportation system for its nuclear translocation and activity and that taxanes inhibit AR signaling downstream of microtubule stabilization. Furthermore, we identified that the AR hinge region mediates binding to MTs; this region is present in ARv567, but missing from ARv7. Thus, the ARv7 does not bind MTs, its nuclear localization is not affected by taxane treatment or dynein motor complex disruption while its expression confers taxane resistance in vivo. Collectively these data suggest that ARv7 confers resistance to both ADT and taxanes, which represent the most important therapeutic modalities in CRPC. To this end, elucidation of the molecular mechanism of ARv7 nuclear translocation is extremely critical for the design of new targeted therapies to treat more than 60% of mCRPC patients who do not respond to current treatment options. To monitor the dynamics of ARv nuclear localization, we used fluorescence recovery after photobleaching (FRAP) assay and measured the time kinetics of ARv nuclear translocation. Our results indicated that ARv7 translocates to the nucleus significantly faster as compared to AR-wt and ARv567. In addition, since AR-wt utilizes the importin-α/β to translocate to the nucleus we used importazole, a tool compound blocking importin-β activity, and measured the dynamics of ARv translocation. We found that importazole was effective in inhibiting AR-wt and ARv567 nuclear import, but had no such effect on ARv7 nuclear accumulation. Currently, we are investigating the novel molecular mechanism of ARv7 nuclear translocation including the involvement of Ran-GTPase and the identification of the putative non-canonical nuclear localization signal. Overall, our data demonstrate that ARv7 utilizes a different nuclear translocation mechanism as compared with AR-wt and ARv567. Elucidation of this mechanism will be invaluable in order to design an alternative therapeutic modality to halt AR signaling which eventually will open new avenues to treat large subset of mCRPC patients who show resistance to current treatments. Citation Format: Seaho Kim, Luigi Portella, Paraskevi Giannakakou. Distinct nuclear translocation mechanism of androgen receptor variant ARv7. [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 3451. doi:10.1158/1538-7445.AM2015-3451