Reza Bayat Mokhtari

Metronomic Oral Topotecan with Pazopanib is an Active Antiangiogenic Regimen in Mouse Models of Aggressive Pediatric Solid Tumor

Purpose: Low dose metronomic (LDM) chemotherapy, combined with VEGF signaling pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth in many adult preclinical cancer models. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, a VEGF receptor inhibitor, in three pediatric extracranial solid tumor mouse models. Experimental Design: In vitro dose–response study of topotecan and pazopanib was conducted on several neuroblastoma, osteosarcoma, and rhabdomyosarcoma cell lines. In vivo antitumor efficacies of the LDM topotecan and pazopanib as single agents and in combination were tested on 4 subcutaneous xenograft models and on 2 neuroblastoma metastatic models. Circulating angiogenic factors such as circulating endothelial cells (CEC), circulating endothelial pro genitor cells (CEP), and microvessel densities were used as surrogate biomarker markers of antiangiogenic activity. Results: In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, combination of topotecan + pazopanib (TP + PZ) showed significant antitumor activity and significant enhancement in survival compared with the respective single agents in all models. Reductions in viable CEP and/or CEC levels and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. Pharmacokinetic studies of both drugs did not reveal any drug–drug interaction. Conclusion: Metronomic administration of TP + PZ showed a statistically significant antitumor activity compared with respective single agents in pediatric tumor mouse models and represent a valid option as a maintenance therapy in aggressive pediatric solid tumors. Clin Cancer Res; 17(17); 5656–67. ©2011 AACR.

Combination therapy in combating cancer

Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-cancer drugs enhances efficacy compared to the mono-therapy approach because it targets key pathways in a characteristically synergistic or an additive manner. This approach potentially reduces drug resistance, while simultaneously providing therapeutic anti-cancer benefits, such as reducing tumour growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. The 5-year survival rates for most metastatic cancers are still quite low, and the process of developing a new anti-cancer drug is costly and extremely time-consuming. Therefore, new strategies that target the survival pathways that provide efficient and effective results at an affordable cost are being considered. One such approach incorporates repurposing therapeutic agents initially used for the treatment of different diseases other than cancer. This approach is effective primarily when the FDA-approved agent targets similar pathways found in cancer. Because one of the drugs used in combination therapy is already FDA-approved, overall costs of combination therapy research are reduced. This increases cost efficiency of therapy, thereby benefiting the “medically underserved”. In addition, an approach that combines repurposed pharmaceutical agents with other therapeutics has shown promising results in mitigating tumour burden. In this systematic review, we discuss important pathways commonly targeted in cancer therapy. Furthermore, we also review important repurposed or primary anti-cancer agents that have gained popularity in clinical trials and research since 2012.

Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial‐to‐mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E‐cadherin and ZO‐1 and increased expression of N‐cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC‐0449, and after knockdown by Shh‐siRNA, and led to reversal of the EMT phenotype. The results with HTB‐9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF‐β1 treated HTB‐9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF‐β1 they did not exhibit tumorigenicity. The TGF‐β1 treated HTB‐9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF‐β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki‐67, Shh, Gli2, and N‐cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF‐β1‐induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF‐β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.

Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines

BackgroundBronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids.MethodsIn vitro studies were used for the dose–response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase).ResultsBoth compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0–80 μM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content.ConclusionsThe combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

Circulating endothelial cells and circulating endothelial precursor cells in patients with osteosarcoma

Circulating endothelial cells (CECs) have been detected at increased numbers in patients with solid cancers. CECs have not been systematically evaluated in patients with osteosarcoma.

Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN).

BackgroundCurrent chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects.ObjectiveThe acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines.Materials and MethodsWe assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo.ResultsCombination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program.ConclusionWe suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.

Carbonic Anhydrase II Mediates Malignant Behavior of Pulmonary Neuroendocrine Tumors

In normal lung, the predominant cytoplasmic carbonic anhydrase (CA) isozyme (CAII) is highly expressed in amine- and peptide-producing pulmonary neuroendocrine cells where its role involves CO2 sensing. Here, we report robust cytoplasmic expression of CAII by immunohistochemistry in the tumor cells of different native neuroendocrine tumor (NET) types, including typical and atypical carcinoids and small-cell lung carcinomas, and in NET and non-NET tumor cell lines. Because, in both pulmonary neuroendocrine cell and related NETs, the hypercapnia-induced secretion of bioactive serotonin (5-hydroxytryptamine) is mediated by CAII, we investigated the role of CAII in the biological behavior of carcinoid cell line H727 and the type II cell-derived A549 using both in vitro clonogenicity and in vivo xenograft model. We show that short hairpin RNA-mediated down-regulation of CAII resulted in significant reduction in clonogenicity of H727 and A549 cells in vitro, and marked suppression of tumor growth in vivo. CAII-short hairpin RNA cell-derived xenografts showed significantly reduced mitosis (phosphohistone H3 marker) and proliferation associated antigen Ki-67 (Ki67 marker), and significantly increased apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Using an apoptosis gene array, we found no association with caspases 3 and 8, but with a novel association of CAII-mediated apoptosis with specific mitochondrial apoptosis-associated proteins. Furthermore, these xenografts showed a significantly reduced vascularization (CD31 marker). Thus, CAII may play a critical role in NET lung tumor growth, angiogenesis, and survival, possibly via 5-hydroxytryptamine, known to drive autocrine tumor growth. As such, CAII is a potential therapeutic target for the difficult-to-treat lung NETs.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24–2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%–100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non–small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencings clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 24(15); 3539–49. ©2018 AACR.

Manganese-enhanced MRI of minimally gadolinium-enhancing breast tumors

To investigate the potential of manganese (Mn)‐enhanced MRI for sensitive detection and delineation of tumors that demonstrate little enhancement on Gd‐DTPA.