Ani Arakelian

A non–RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo

Purpose: Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to α5β1 and αvβ3 in vitro, to block breast cancer growth and metastasis. Experimental design: MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05–1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. Result: Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. Conclusion: These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer. [Mol Cancer Ther 2006;5(9):2271–80]

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA‐uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136‐143). This 8‐mer capped peptide (Å6) inhibited breast cancer cell invasion and endothelial cell migration in a dose‐dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of Å6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from Å6‐treated animals. Å6 treatment also resulted in a decrease in factor VIII‐positive tumor microvessel hot‐spots. These results identify a new epitope in uPA that is involved in the uPA‐uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.—Guo, Y., Higazi, A. A., Arakelian, A., Sachais, B. S., Cines, D., Goldfarb, R. H., Jones, T. R., Kwaan, H., Mazar, A. P., Rabbani, S. A. A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 14, 1400–1410 (2000)

SKI-606 (Bosutinib) Blocks Prostate Cancer Invasion, Growth, and Metastasis In vitro and In vivo through Regulation of Genes Involved in Cancer Growth and Skeletal Metastasis

In the current study, we have examined the efficacy of a Src/Abl kinase inhibitor SKI-606 (Bosutinib) for its effect on prostate cancer growth and skeletal metastasis. Treatment of highly invasive human prostate cancer cells PC-3 and DU-145 with different doses of SKI-606 decreased Src activation, cell proliferation, migration, and invasion as determined by Matrigel Boyden chamber invasion assay. For in vivo studies, PC-3 cells were inoculated through s.c. or i.t. route into male BALB/c nu/nu or Fox Chase severe combined immunodeficient mice, respectively. Experimental animals treated with SKI-606 developed tumors of a significantly smaller volume and a significant decrease (50%) in experimental skeletal lesion area. A marked increase (32%) in bone volume to tumor volume ratio was also seen by micro–computed tomography analysis of tibias from control and experimental groups of animals. Western blot analysis showed the ability of SKI-606 to significantly decrease the phosphorylation of signaling molecules (AKT, mitogen-activated protein kinase, focal adhesion kinase) and the expression of tumor progression–associated genes uPAR, MMP-2, MMP-9, N-cadherin, fibronectin, BMP-2 (bone morphogenetic protein 2), BMP-6 (bone morphogenetic protein 6), IL-8 (interleukin 8), and TGF-β (transforming growth factor β) in prostate cancer cells. SKI-606 is currently in clinical trials for breast cancer and chronic myelogenous leukemia. Results from these studies provide convincing evidence for evaluating its efficacy in prostate cancer patients. Mol Cancer Ther; 9(5); 1147–57. ©2010 AACR.

A Synthetic 15-mer Peptide (PCK3145) Derived from Prostate Secretory Protein Can Reduce Tumor Growth, Experimental Skeletal Metastases, and Malignancy-Associated Hypercalcemia

In previous studies, we have shown that prostate secretory protein (PSP-94) can reduce prostate cancer growth in vivo. In the current study, we identified the amino acid sequence of PSP-94 that is required for eliciting this response. For these studies, we used rat prostate cancer Mat Ly Lu cells overexpressing parathyroid hormone-related protein (PTHrP), which is the main pathogenetic factor responsible for hypercalcemia of malignancy. Synthetic peptides corresponding to amino acids 7–21 (PCK721), 31–45 (PCK3145), and 76–94 (PCK7694) of PSP-94 were synthesized. Only PCK3145 showed a significant reduction in tumor cell proliferation. For in vivo studies, syngenic male Copenhagen rats were inoculated s.c. with Mat Ly Lu cells overexpressing PTHrP into the right flank or into the left ventricle via intracardiac injection, which results in experimental metastases to the lumbar vertebrae causing hind-limb paralysis. Animals were infused with different doses (1, 10, and 100 μg/kg/day) of peptides for 15 days, and the effect of these treatments on tumor volume, skeletal metastases, or development of hind-limb paralysis was determined. Treatment with PCK3145 resulted in a dose-dependent decrease in tumor volume and delay in the development of skeletal metastases. Bone histomorphometry showed that after intracardiac inoculation of tumor cells, the highest dose of PCK3145 (100 μg/kg/day) resulted in reducing skeletal tumor burden, which delayed the development of hind-limb paralysis. Treatment with PCK3145 led to reduction of plasma calcium and PTHrP levels and a significant decrease in PTHrP levels in the primary tumors and in vertebrae of experimental animals. These effects of PCK3145 were due to its ability to promote tumor cell apoptosis. Collectively, the results of these studies have demonstrated the ability of a small peptide derived from PSP-94 to reduce tumor volume and experimental skeletal metastases—results that will be highly beneficial in the continued development of this peptide as a novel therapeutic agent for patients with hormone refractory, late-stage prostate cancer.

Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion.

IntroductionDeregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFβ) in breast cancer progression.MethodsCyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion.ResultsWe found TGFβ to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFβ-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFβ-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFβ promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21.ConclusionsThus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFβ-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

IntroductionTumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.MethodsA mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.ResultsWe found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.ConclusionsTogether these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Genome-Wide Study of Hypomethylated and Induced Genes in Patients with Liver Cancer Unravels Novel Anticancer Targets

Purpose: We utilized whole-genome mapping of promoters that are activated by DNA hypomethylation in hepatocellular carcinoma (HCC) clinical samples to shortlist novel targets for anticancer therapeutics. We provide a proof of principle of this approach by testing six genes short-listed in our screen for their essential role in cancer growth and invasiveness. Experimental Design: We used siRNA- or shRNA-mediated depletion to determine whether inhibition of these genes would reduce human tumor xenograft growth in mice as well as cell viability, anchorage-independent growth, invasive capacities, and state of activity of nodal signaling pathways in liver, breast, and bladder cancer cell lines. Results: Depletion of EXOSC4, RNMT, SENP6, WBSCR22, RASAL2, and NENF effectively and specifically inhibits cancer cell growth and cell invasive capacities in different types of cancer, but, remarkably, there is no effect on normal cell growth, suggesting a ubiquitous causal role for these genes in driving cancer growth and metastasis. Depletion of RASAL2 and NENF in vitro reduces their growth as explants in vivo in mice. RASAL2 and NENF depletion interferes with AKT, WNT, and MAPK signaling pathways as well as regulation of epigenetic proteins that were previously demonstrated to drive cancer growth and metastasis. Conclusion: Our results prove that genes that are hypomethylated and induced in tumors are candidate targets for anticancer therapeutics in multiple cancer cell types. Because these genes are particularly activated in cancer, they constitute a group of targets for specific pharmacologic inhibitors of cancer and cancer metastasis. Clin Cancer Res; 20(12); 3118–32. ©2014 AACR.

Effect of Herceptin on the development and progression of skeletal metastases in a xenograft model of human breast cancer

We examined the effects of Herceptin, a bioengineered monoclonal antibody directed against Her-2/neu oncogene on skeletal metastasis using a xenograft model of breast cancer. Treatment of Her-2 overexpressing human breast cancer cells BT-474 with Herceptin caused a dose-dependent decrease in cell proliferation. In in vivo studies, BT-474 cells (1 × 105) were injected into the left ventricle of female BALB/c nu/nu mice. Intraperitoneal (i.p.) infusion of Herceptin (1 mg/kg twice a week for 5 weeks) from the day of tumor cell inoculation or at the time of radiologically detectable skeletal metastasis either slowed the development or prevented the progression of skeletal metastasis as compared to control groups of animals receiving nonspecific IgG. Bone histological analysis of long bones showed the ability of Herceptin to reduce the ratio of tumor volume to bone volume as well as mitotic index, effects that were more pronounced when Herceptin treatment was initiated from the day of tumor cell inoculation. While immunohistochemical analysis of long bones showed no difference in the production of Her-2, phosphorylated (P) Her-2 and MAPK, a significantly lower level of P-MAPK was seen in bones of Herceptin treated animals. These studies demonstrate the ability of Herceptin to inhibit the development and abrogate the progression of skeletal metastases associated with breast cancer by blocking the HER-2-mediated signaling pathways.

LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo.

Prostate cancer (PCa) is a common hormone‐dependent malignancy associated with the development of skeletal metastases. This is due to the increased expression of a number of growth factors, cytokines, and proteases which collectively drive the metastatic cascade in general and increased propensity to develop skeletal metastasis in particular. While a number of signaling pathways have been implicated in PCa progression, the highly complex wnt/β‐catenin pathway is unique due to its ability to regulate gene expression, cell invasion, migration, survival, proliferation, and differentiation to contribute in the initiation and progression of PCa. Members of the wnt family bind to the Frizzle proteins or lipoprotein‐related receptor proteins 5, 6 (LRP5, ‐6) to activate this key pathway. In the current study, we have investigated the role of wnt/β‐catenin pathway in PCa progression, skeletal metastasis, and gene expression using the dominant negative plasmid of LRP5 (DN‐LRP5) and human PCa cells PC‐3. Inactivation of LRP5 resulted in mesenchymal to epithelial shift, lack of translocation of β‐catenin to cell surface, increased tumor cell proliferation, decreased colony formation, migration and invasion in vitro. These effects were attributed to decreased expression of pro‐invasive and pro‐metastatic genes. In in vivo studies, PC‐3‐DN‐LRP5 cells developed significantly smaller tumors and a marked decrease in skeletal lesion area and number as determined by X‐ray, micro (μ) CT and histological analysis. Collectively results from these studies demonstrate the dominant role of this key pathway in PCa growth and skeletal metastasis and its potential as a viable therapeutic target.

S‐adenosylmethionine blocks osteosarcoma cells proliferation and invasion in vitro and tumor metastasis in vivo: therapeutic and diagnostic clinical applications

Osteosarcoma (OS) is an aggressive and highly metastatic form of primary bone cancer affecting young children and adults. Previous studies have shown that hypomethylation of critical genes is driving metastasis. Here, we examine whether hypermethylation treatment can block OS growth and pulmonary metastasis. Human OS cells LM‐7 and MG‐63 were treated with the ubiquitous methyl donor S‐adenosylmethionine (SAM) or its inactive analog S‐adenosylhomocystine (SAH) as control. Treatment with SAM resulted in a dose‐dependent inhibition of tumor cell proliferation, invasion, cell migration, and cell cycle characteristics. Inoculation of cells treated with 150 μmol/L SAM for 6 days into tibia or via intravenous route into Fox Chase severe combined immune deficient (SCID) mice resulted in the development of significantly smaller skeletal lesions and a marked reduction in pulmonary metastasis as compared to control groups. Epigenome wide association studies (EWAS) showed differential methylation of several genes involved in OS progression and prominent signaling pathways implicated in bone formation, wound healing, and tumor progression in SAM‐treated LM‐7 cells. Real‐time polymerase chain reaction (qPCR) analysis confirmed that SAM treatment blocked the expression of several prometastatic genes and additional genes identified by EWAS analysis. Immunohistochemical analysis of normal human bone and tissue array from OS patients showed significantly high levels of expression of one of the identified gene platelet‐derived growth factor alpha (PDGFA). These studies provide a possible mechanism for the role of DNA demethylation in the development and metastasis of OS to provide a rationale for the use of hypermethylation therapy for OS patients and identify new targets for monitoring OS development and progression.