Giacomo Canesin

Galectin-3 expression is associated with bladder cancer progression and clinical outcome

Galectin-3 belongs to a family of carbohydrate-binding proteins whose function is not fully characterized. However, it is believed to play a role in adhesion, proliferation and apoptosis in solid tumors. We aimed at investigating galectin-3 expression in bladder cancer. Galectin-3 expression was assessed by transcript profiling (U133A arrays) in a series or frozen bladder tumors (n = 105). Immunohistochemistry was performed on tissue arrays containing bladder tumors (n = 389) to evaluate associations of protein expression patterns of galectin-3 with proliferation (Ki67), apoptosis (apopdetek), bcl-2, and clinicopathologic variables. Galectin-3 protein levels were then quantified in 160 urinary specimens of bladder cancer patients and controls by enzymeimmunoanalysis. Galectin-3 gene expression levels increased in invasive tumours as compared with non-muscle invasive lesions (p = 0.001) and were associated with poor survival in patients with advanced disease (p = 0.03). Protein expression patterns also correlated galectin-3 with tumor stage (p < 0.001), grade (p = 0.03), Ki67 and apopdetek (p < 0.001), and overall survival in patients with T1G3 tumors (p < 0.001). Furthermore, galectin-3 urinary levels segregated bladder cancer patients from controls with high diagnostic accuracy (AUC = 0.7). Independent series of bladder tumors showed that transcript and protein levels of galectin-3 were differentially expressed along bladder cancer progression. Urinary protein levels served to identify bladder cancer patients. These observations suggest a role for galectin-3 as a biomarker for bladder cancer diagnostics, staging, and outcome prognosis.

Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization

Epithelial–mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.

LOXL2 catalytically inactive mutants mediate epithelial-to-mesenchymal transition

Summary Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT) by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

The STAT3 Inhibitor Galiellalactone Effectively Reduces Tumor Growth and Metastatic Spread in an Orthotopic Xenograft Mouse Model of Prostate Cancer

UNLABELLED Signal transducer and activator of transcription 3 (STAT3) is known to be involved in the progression of prostate cancer (PCa) and is a key factor in drug resistance and tumor immunoescape. As a result, it represents a promising target for PCa therapy. We studied the effects of the STAT3 inhibitor galiellalactone (GL) on tumor growth and metastatic spread in vitro and in vivo. The effect of GL on cell viability, apoptosis, and invasion was studied in vitro using androgen-independent DU145 and DU145-Luc cell lines. For in vivo studies, mice were injected orthotopically with DU145-Luc cells and treated with daily intraperitoneal injections of GL for 6 wk. GL significantly reduced the growth of the primary tumor and the metastatic spread of PCa cells to regional and distal lymph nodes in vivo. Treatment with GL also resulted in decreased cell proliferation and increased apoptosis compared with controls. In vitro, GL reduces the viability and invasive abilities of DU145-Luc cells and induces apoptosis. Our results showed that tumor growth and early metastatic dissemination of PCa can be significantly reduced by GL, indicating its potential use as a therapeutic compound in advanced metastatic PCa. PATIENT SUMMARY In this study, we tested the STAT3 inhibitor galiellalactone (GL) in an animal model of PCa. We found that mice treated with GL had smaller primary tumors and decreased lymph node metastases compared with mice treated with vehicle. GL has potential for treating advanced metastatic PCa.

Circulating Tumor Cells as a Marker for Progression-free Survival in Metastatic Castration-naïve Prostate Cancer

Analysis of circulating tumor cells (CTC) is a promising prognostic marker in castration‐resistant prostate cancer (CRPC). The aim of this study was to investigate CTC detection and phenotyping as prognostic biomarkers for response to primary androgen deprivation therapy (ADT) of metastatic prostate cancer (PC).

Treatment with the WNT5A-mimicking peptide Foxy-5 effectively reduces the metastatic spread of WNT5A-low prostate cancer cells in an orthotopic mouse model

Prostate cancer patients with high WNT5A expression in their tumors have been shown to have more favorable prognosis than those with low WNT5A expression. This suggests that reconstitution of Wnt5a in low WNT5A-expressing tumors might be an attractive therapeutic approach. To explore this idea, we have in the present study used Foxy-5, a WNT5A mimicking peptide, to investigate its impact on primary tumor and metastasis in vivo and on prostate cancer cell viability, apoptosis and invasion in vitro. We used an in vivo orthotopic xenograft mouse model with metastatic luciferase-labeled WNT5A-low DU145 cells and metastatic luciferase-labeled WNT5A-high PC3prostate cancer cells. We provide here the first evidence that Foxy-5 significantly inhibits the initial metastatic dissemination of tumor cells to regional and distal lymph nodes by 90% and 75%, respectively. Importantly, this effect was seen only with the WNT5A-low DU145 cells and not with the WNT5A-high PC3 cells. The inhibiting effect in the DU145-based model occurred despite the fact that no effects were observed on primary tumor growth, apoptosis or proliferation. These findings are consistent with and supported by the in vitro data, where Foxy-5 specifically targets invasion without affecting apoptosis or viability of WNT5A-low prostate cancer cells. To conclude, our data indicate that the WNT5A-mimicking peptide Foxy-5, which has been recently used in a phase 1 clinical trial, is an attractive candidate for complimentary anti-metastatic treatment of prostate cancer patients with tumors exhibiting absent or low WNT5A expression.

Preclinical Characterization of 3β-(N-Acetyl l-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (GPA512), a Prodrug of a Direct STAT3 Inhibitor for the Treatment of Prostate Cancer

The transcription factor STAT3 is a potential target for the treatment of castration-resistant prostate cancer. Galiellalactone (1), a direct inhibitor of STAT3, prevents the transcription of STAT3 regulated genes. In this study we characterized 6 (GPA512, Johansson , M. ; Sterner , O. Patent WO 2015/132396 A1, 2015 ), a prodrug of 1. In vitro studies showed that 6 is rapidly converted to 1 in plasma and is stable in a buffer solution. The pharmacokinetics of 6 following a single oral dose indicated that the prodrug was rapidly absorbed and converted to 1 with a tmax of 15 min. Oral administration of 6 in mice increased the plasma exposure of the active parent compound 20-fold compared to when 1 was dosed orally. 6 treated mice bearing DU145 xenograft tumors had significantly reduced tumor growth compared to untreated mice. The favorable druglike properties and safety profile of 6 warrant further studies of 6 for the treatment of castration-resistant prostate cancer.

Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis

Cartilage oligomeric matrix protein (COMP) was recently implicated in the progression of breast cancer. Immunostaining of 342 prostate cancer specimens in tissue microarrays showed that COMP expression is not breast cancer-specific but also occurs in prostate cancer. The expression of COMP in prostate cancer cells correlated with a more aggressive disease with faster recurrence. Subcutaneous xenografts in immunodeficient mice showed that the prostate cancer cell line DU145 overexpressing COMP formed larger tumors in vivo as compared to mock-transfected cells. Purified COMP bound to and enhanced the invasion of DU145 cells in vitro in an integrin-dependent manner. In addition, intracellular COMP expression interfered with cellular metabolism by causing a decreased level of oxidative phosphorylation with a concurrent upregulation of lactate production (Warburg effect). Further, expression of COMP protected cells from induction of apoptosis via several pathways. The effect of COMP on metabolism and apoptosis induction was dependent on the ability of COMP to disrupt intracellular Ca2+ signalling by preventing Ca2+ release from the endoplasmic reticulum. In conclusion, COMP is a potent driver of the progression of prostate cancer, acting in an anti-apoptotic fashion by interfering with the Ca2+ homeostasis of cancer cells.

Abstract A040: Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone

Introduction and Objectives: Cancer stem-like cells (CSCs) represent a small subpopulation of largely quiescent cells that reside within tumors. Several studies have demonstrated that this population is more resistant to current therapies and is therefore directly responsible for tumor recurrence. Recent evidence suggests that the transcription factor STAT3 is crucial to the survival of stem cells in prostate cancer (PCa). Thus, inhibition of activated STAT3 (pSTAT3) is a valid strategy to selectively target PCa CSCs. We have previously shown that pSTAT3 blockade, by galielallactone (GL), not only reduces proliferation and induces apoptosis of prostate cancer cells in vitro, but it also inhibits the growth of prostate tumors and the metastatic spread to regional and distal lymph nodes, in vivo. Here we performed experiments aimed at studying the effect of the STAT3 inhibitor, GL, on PCa CSCs, as a promising therapeutic approach for prostate cancer patients. Materials and Methods: The expression of stem, basal, and luminal cell surface markers (CD133/CD44/CD24) was analyzed by FACS on DU145 and PC3 cells following treatment with GL. DU145 cells were sorted, based on the expression of the stem cell surface marker CD133, and then plated for further analysis. Expression of pSTAT3 was studied by IHC, on sorted cells, and the effect of GL on clonogenic recovery was determined. Furthermore, the effect of GL on sphere formation efficiency was studied on the CD133 population. The compound galiellalactone was obtained from Glactone Pharma (Sweden). Results: Our results show that the CSCs population (CD133+/CD44+) expresses high levels of activated pSTAT3 compared to the CD44+/CD24+ cell population. Treatment with GL decreased the number of CSCs in DU145 cells after 48h, while the number of CD44+/CD24+ cells was not significantly affected. Importantly, treatment with GL had no effect on CD133+/CD44+ or CD44+/CD24+ populations in PC3 cells, which do not express pSTAT3. Treatment with GL significantly reduced the ability of CSCs to form colonies and to generate prostate-derived spheres. Interestingly, the CSCs population (CD133+) was more sensitive to GL in terms of colony- and sphere-forming efficiency compared to the CD133- population. Conclusion: This study demonstrates that the STAT3 inhibitor galiellalactone can specifically target the prostate cancer stem-like cell population in vitro, implying that pSTAT3 inhibition by GL could represent a valid therapeutic strategy to antagonize CSCs in human prostate cancer. Future experiments will be aimed at validating these data and relating the clonogenic inhibition to the effects of pSTAT3 blockade by GL on tumor initiation by prostate cancer stem-like cells in vivo. Citation Format: Giacomo Canesin, Anne T. Collins, Rebecka Hellsten, Norman J. Maitland, Anders Bjartell. Targeting the prostate cancer stem cell niche with the STAT3 inhibitor galiellalactone [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A040.

Abstract C179: Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer

Background: The transcription factor STAT3 is a promising target for the treatment of castration-resistant prostate cancer (CRPC) as STAT3 is implicated in drug resistance and castration resistance as well as metastatic spread, tumor growth and immune escape. Galiellalactone (GL) is a direct inhibitor of STAT3 that prevents DNA binding, inhibits proliferation of prostate cancer cells expressing active STAT3 and induces apoptosis by down-regulation of STAT3 activated genes. In this study we aimed to characterize a prodrug of GL, GPA512, with improved drug-like properties and to demonstrate its effect on tumor growth in a xenograft model of prostate cancer following oral administration. Methods: Stability studies of prodrugs based on GL were performed in 0.1 M PBS buffer (pH 7.4) and in plasma at 37°C. In vitro efficacy of prodrugs was studied by WST-1 proliferation assay in DU145 prostate cancer cells expressing active STAT3. The systemic exposure of GL in mice was studied following a single oral dose of GPA512 or GL (both 10 mg/kg). The plasma concentrations of GL were determined by LC-MS/MS. For the xenograft study NMRI-nude male mice were inoculated subcutaneously with DU145 cells and once tumors were established the mice were divided in two groups with ten mice in each. Mice were treated orally with 40 mg/kg GPA512 daily five times/week for four weeks. Tumor growth was measured by caliper and at the end of the study tumors were harvested for subsequent analyses using immunohistochemistry and mRNA expression analysis. Results: In vitro studies showed that the prodrug GPA512 is rapidly converted to GL in plasma and that GPA512 is stable in buffer solution and has similar inhibitory effects on proliferation as GL on DU145 cells. The pharmacokinetics of GPA512 following a single oral dose indicated that the compound was rapidly absorbed and converted to GL with a tmax of 15 min. Oral administration of GPA512 in mice increased the plasma exposure (AUC) of the active parent compound 20-fold compared to when GL was dosed orally. GPA512 treated mice bearing subcutaneous DU145 tumors had significantly smaller tumors compared to mice treated with vehicle. No adverse effects or weight loss were observed. Analysis of tumors showed decreased cell proliferation and increased amount of apoptotic cells in GPA512 treated mice compared to control. The mRNA expression of STAT3 regulated anti-apoptotic gene MCl-1 was significantly reduced by GPA512 treatment. Conclusions: The drug-like properties and safety profile of the prodrug GPA512 and galiellalactone9s direct inhibition of STAT3, warrant further studies of GPA512 as a drug candidate for treatment of patients with CRPC. Citation Format: Rebecka Hellsten, Zilma Escobar, Anders Bjartell, Giacomo Canesin, Susan Evans Axelsson, Olov Sterner, Martin Johansson. Preclinical characterization of GPA512: A prodrug of a direct STAT3 inhibitor for the treatment of prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C179.