S-adenosylmethionine in combination with decitabine shows enhanced anti-cancer effects in repressing breast cancer growth and metastasis.

Abnormal DNA methylation orchestrates many of the cancer-related gene expression irregularities such as the inactivation of tumour suppressor genes through hypermethylation as well as activation of prometastatic genes through hypomethylation. The fact that DNA methylation abnormalities can be chemically reversed positions the DNA methylation machinery as an attractive target for anti-cancer drug development. However, although in vitro studies suggested that targeting concordantly hypo- and hypermethylation is of benefit in suppressing both oncogenic and prometastatic functions of breast cancer cells, this has never been tested in a therapeutic setting in vivo. In this context, we investigated the combined therapeutic effects of an approved nutraceutical agent S-adenosylmethionine (SAM) and FDA-approved hypomethylating agent decitabine using the MDA-MB-231 xenograft model of breast cancer and found a pronounced reduction in mammary tumour volume and lung metastasis compared to the animals in the control and monotherapy treatment arms. Immunohistochemical assessment of the primary breast tumours showed a significantly reduced expression of proliferation (Ki-67) and angiogenesis (CD31) markers following combination therapy as compared to the control group. Global transcriptome and methylome analyses have revealed that the combination therapy regulates genes from several key cancer-related pathways that are abnormally expressed in breast tumours. To our knowledge, this is the first preclinical study demonstrating the anti-cancer therapeutic potential of using a combination of methylating (SAM) and demethylating agent (decitabine) in vivo. Results from this study provide a molecularly founded rationale for clinically testing a combination of agents targeting the epigenome to reduce the morbidity and mortality from breast cancer.

DNA methylation signatures of Prostate Cancer in peripheral T-cells.

BACKGROUND: Prostate Cancer (PCa) is the second most common cancer in men where advancements have been made for early detection using imaging techniques, however these are limited by lesion size. Immune surveillance has emerged as an effective approach for early detection and to monitor disease progression. In recent studies, we have shown that host peripheral blood immune cells undergo changes in DNA methylation in liver and breast cancer. METHODS: In the current study, we examined the DNA methylation status of peripheral blood T cells of men with positive biopsy for PCa versus men with negative biopsy having benign prostate tissue, defined as controls. T cells DNA was isolated and subjected to Illumina Infinium methylation EPIC array and validated using Illumina amplicon sequencing and pyrosequencing platforms. RESULTS: Differential methylation of 449 CG sites between control and PCa T cell DNA showed a correlation with Gleason score (p < 0.05). Two hundred twenty-three differentially methylated CGs between control and PCa (Ƨ +/- 10%, p < 0.05), were enriched in pathways involved in immune surveillance system. Three CGs which were found differentially methylated following DMP (Differentially methylated probes) analysis of ChAMP remained significant after BH (Benjamini-Hochberg) correction, of which, 2 CGs were validated. Predictive ability of combination of these 3 CGs (polygenic methylation score, PMS) to detect PCa had high sensitivity, specificity and overall accuracy. PMS also showed strong positive correlation with Gleason score and tumor volume of PCa patients. CONCLUSIONS: Results from the current study provide for the first-time a potential role of DNA methylation changes in peripheral T cells in PCa. This non-invasive methodology may allow for early intervention and stratification of patients into different prognostic groups to reduce PCa associated morbidity from repeat invasive prostate biopsies and design therapeutic strategy to reduce PCa associated mortality.

DNA methylation signatures of breast cancer in peripheral T-cells.

BACKGROUND: Immune surveillance acts as a defense mechanism in cancer, and its disruption is involved in cancer progression. DNA methylation reflects the phenotypic identity of cells and recent data suggested that DNA methylation profiles of T cells and peripheral blood mononuclear cells (PBMC) are altered in cancer progression. METHODS: We enrolled 19 females with stage 1 and 2, nine with stage 3 and 4 and 9 age matched healthy women. T cells were isolated from peripheral blood and extracted DNA was subjected to Illumina 450 K DNA methylation array analysis. Raw data was analyzed by BMIQ, ChAMP and ComBat followed by validation of identified genes by pyrosequencing. RESULTS: Analysis of data revealed ~ 10,000 sites that correlated with breast cancer progression and established a list of 89 CG sites that were highly correlated (p < 0.01, r > 0.7, r < - 0.7) with breast cancer progression. The vast majority of these sites were hypomethylated and enriched in genes with functions in the immune system. CONCLUSIONS: The study points to the possibility of using DNA methylation signatures as a noninvasive method for early detection of breast cancer and its progression which need to be tested in clinical studies.

Synergistic effects of combined DNA methyltransferase inhibition and MBD2 depletion on breast cancer cells; MBD2 depletion blocks 5-aza-2'-deoxycytidine-triggered invasiveness.

5-Aza-2'-deoxycytidine (5-azaCdR) not only inhibits growth of non-invasive breast cancer cells but also increases their invasiveness through induction of pro-metastatic genes. Methylated DNA binding protein 2 (MBD2) is involved in silencing methylated tumor suppressor genes as well as activation of pro-metastatic genes. In this study, we show that a combination of MBD2 depletion and DNA methyltransferases (DNMT) inhibition in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumor growth arrest on one hand, while inhibiting invasiveness triggered by 5-azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by combination of MBD2 and DNMT inhibitors.

Methyl-CpG binding domain protein 2 (Mbd2) drives breast cancer progression through the modulation of epithelial-to-mesenchymal transition.

Methyl-CpG-binding domain protein 2 (Mbd2), a reader of DNA methylation, has been implicated in different types of malignancies, including breast cancer. However, the exact role of Mbd2 in various stages of breast cancer growth and progression in vivo has not been determined. To test whether Mbd2 plays a causal role in mammary tumor growth and metastasis, we performed genetic knockout (KO) of Mbd2 in MMTV-PyMT transgenic mice and compared mammary tumor progression kinetics between the wild-type (PyMT-Mbd2+/+) and KO (PyMT-Mbd2-/-) groups. Our results demonstrated that deletion of Mbd2 in PyMT mice impedes primary tumor growth and lung metastasis at the experimental endpoint (postnatal week 20). Transcriptomic and proteomic analyses of primary tumors revealed that Mbd2 deletion abrogates the expression of several key determinants involved in epithelial-to-mesenchymal transition, such as neural cadherin (N-cadherin) and osteopontin. Importantly, loss of the Mbd2 gene impairs the activation of the PI3K/AKT pathway, which is required for PyMT-mediated oncogenic transformation, growth, and survival of breast tumor cells. Taken together, the results of this study provide a rationale for further development of epigenetic therapies targeting Mbd2 to inhibit the progression of breast cancer.

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

INTRODUCTION: Deregulation 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 (TGFß) in breast cancer progression. METHODS: Cyclins 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. RESULTS: We found TGFß to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFß-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFß-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFß 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. CONCLUSIONS: Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFß-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 TGFß-mediated breast cancer cell migration and invasion.

INTRODUCTION: Tumor 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 (TGFß)-mediated breast cancer cell migration and invasion. METHODS: A 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 TGFß and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFß transcriptional activity was measured by a TGFß/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFß 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 TGFß/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. RESULTS: We 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 TGFß. p21 and p/CAF regulates TGFß-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFß/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients. CONCLUSIONS: Together 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.

Co-Targeting Luminal B Breast Cancer with S-Adenosylmethionine and Immune Checkpoint Inhibitor Reduces Primary Tumor Growth and Progression, and Metastasis to Lungs and Bone.

Breast cancer (BCa) is the most prevalent cancer in females and has a high rate of mortality, especially due to increased metastasis to skeletal and non-skeletal sites. Despite the marked clinical accomplishment of immune checkpoint inhibitor (CPI) therapy in patients with several cancers, it has had limited success in luminal subtypes of BCa. Accordingly, recent efforts have focused on combination therapy with CPI, including epigenetic modulators, to increase response rates of CPI in luminal BCa. We have previously shown that S-adenosylmethionine (SAM), the ubiquitous methyl donor, has strong anti-cancer effects in various cancers, including all subtypes of BCa. In the current study, we took a novel approach and examined the effect of CPI alone and in combination with SAM on tumor growth and metastasis in a syngeneic mouse model of luminal B BCa. We showed that SAM decreases cell proliferation, colony-formation (survival), and invasion of luminal B BCa cell lines (Eo771, R221A) in vitro. In in vivo studies, in Eo771 tumor-bearing mice, either SAM or anti-PD-1 antibody treatment alone significantly reduced tumor growth and progression, while the SAM+anti-PD-1 combination treatment had the highest anti-cancer efficacy of all groups. The SAM+anti-PD-1 combination reduced the percentage of animals with lung metastasis, as well as total metastatic lesion area, compared to control. Additionally, the SAM+anti-PD-1 combination significantly reduced the skeletal lesion area and protected tibial integrity to a greater extent than the monotherapies in an Eo771 bone metastasis model. Transcriptome analysis of Eo771 primary tumors revealed significant downregulation of pro-metastatic genes, including Matrix metalloproteinases (MMPs) and related pathways. On the other hand, CD8+ T cell infiltration, CD8+ T cell cytotoxicity (elevated granzymes), and immunostimulatory genes and pathways were significantly upregulated by the combination treatment. The results presented point to a combination of SAM with CPI as a possible treatment for luminal B BCa that should be tested in clinical studies.

An enhanced chemopreventive effect of methyl donor S-adenosylmethionine in combination with 25-hydroxyvitamin D in blocking mammary tumor growth and metastasis.

Therapeutic targeting of metastatic breast cancer still remains a challenge as the tumor cells are highly heterogenous and exploit multiple pathways for their growth and metastatic spread that cannot always be targeted by a single-agent monotherapy regimen. Therefore, a rational approach through simultaneous targeting of several pathways may provide a better anti-cancer therapeutic effect. We tested this hypothesis using a combination of two nutraceutical agents S-adenosylmethionine (SAM) and Vitamin D (Vit. D) prohormone [25-hydroxyvitamin D; '25(OH)D'] that are individually known to exert distinct changes in the expression of genes involved in tumor growth and metastasis. Our results show that both SAM and 25(OH)D monotherapy significantly reduced proliferation and clonogenic survival of a panel of breast cancer cell lines in vitro and inhibited tumor growth, lung metastasis, and breast tumor cell colonization to the skeleton in vivo. However, these effects were significantly more pronounced in the combination setting. RNA-Sequencing revealed that the transcriptomic footprint on key cancer-related signaling pathways is broader in the combination setting than any of the monotherapies. Furthermore, comparison of the differentially expressed genes from our transcriptome analyses with publicly available cancer-related dataset demonstrated that the combination treatment upregulates genes from immune-related pathways that are otherwise downregulated in bone metastasis in vivo. Since SAM and Vit. D are both approved nutraceuticals with known safety profiles, this combination treatment may serve as a novel strategy to reduce breast cancer-associated morbidity and mortality.

Enhanced Anticancer Effect of a Combination of S-adenosylmethionine (SAM) and Immune Checkpoint Inhibitor (ICPi) in a Syngeneic Mouse Model of Advanced Melanoma.

Immune checkpoint inhibitors (ICPi) targeting the PD-1/PD-L1 pathway have shown marked success in patients with advanced melanoma. However, 60-70% of patients fail to respond, warranting a therapeutic intervention that could increase response rates. We and others have shown that S-adenosylmethionine (SAM), a universal methyl donor, has significant anticancer effects in numerous cancers previously; however, its effect on melanoma progression has not been evaluated. Interestingly, SAM was reported to be essential for T cell activation and proliferation and, thus, could potentially cooperate with ICPi and block melanoma progression. In this study, we examined the antitumor effects of SAM and ICPi alone and in combination in a well-established melanoma mouse model wherein syngeneic C57BL/6 mouse were subcutaneously (orthotopic) injected with B16-F1 cells. Treatment of mice with either SAM or anti-PD-1 antibody alone resulted in significant reduction in tumor volumes and weights; effects that were highest in mice treated with a combination of SAM+anti-PD-1. RNA-sequencing analysis of the primary tumors showed numerous differentially expressed genes (DEGs) following treatment with SAM+anti-PD-1, which was shown to downregulate cancer, MAPK, and tyrosine kinase pathways. Indeed, SAM+anti-PD-1 reversed the aberrant expression of some known melanoma genes. Tumor immunophenotyping revealed the SAM+anti-PD-1 combination was significantly more effective than either SAM or anti-PD-1 as the CD8+ T cells had higher activation, proliferation, and cytokine production compared to all other groups. This study shows that the combination of currently approved agents SAM and ICPi can effectively block melanoma via alteration of key genes/pathways implicated in cancer and immune response pathways, providing the rationale for the initiation of clinical trials with SAM and ICPi.

uPAR antibody (huATN-658) and Zometa reduce breast cancer growth and skeletal lesions.

Urokinase plasminogen activator receptor (uPAR) is implicated in tumor growth and metastasis due to its ability to activate latent growth factors, proteases, and different oncogenic signaling pathways upon binding to different ligands. Elevated uPAR expression is correlated with the increased aggressiveness of cancer cells, which led to its credentialing as an attractive diagnostic and therapeutic target in advanced solid cancer. Here, we examine the antitumor effects of a humanized anti-uPAR antibody (huATN-658) alone and in combination with the approved bisphosphonate Zometa (Zoledronic acid) on skeletal lesion through a series of studies in vitro and in vivo. Treatment with huATN-658 or Zometa alone significantly decreased human MDA-MB-231 cell proliferation and invasion in vitro, effects which were more pronounced when huATN-658 was combined with Zometa. In vivo studies demonstrated that huATN-658 treatment significantly reduced MDA-MB-231 primary tumor growth compared with controls. In a model of breast tumor-induced bone disease, huATN-658 and Zometa were equally effective in reducing skeletal lesions. The skeletal lesions were significantly reduced in animals receiving the combination of huATN-658 + Zometa compared with monotherapy treatment. These effects were due to a significant decrease in osteoclastic activity and tumor cell proliferation in the combination treatment group. Transcriptome analysis revealed that combination treatment significantly changes the expression of genes from signaling pathways implicated in tumor progression and bone remodeling. Results from these studies provide a rationale for the continued development of huATN-658 as a monotherapy and in combination with currently approved agents such as Zometa in patients with metastatic breast cancer.

Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications.

DNA hypomethylation coordinately targets various signaling pathways involved in tumor growth and metastasis. At present, there are no approved therapeutic modalities that target hypomethylation. In this regard, we examined the therapeutic plausibility of using universal methyl group donor S-adenosylmethionine (SAM) to block breast cancer development, growth, and metastasis through a series of studies in vitro using two different human breast cancer cell lines (MDA-MB-231 and Hs578T) and in vivo using an MDA-MB-231 xenograft model of breast cancer. We found that SAM treatment caused a significant dose-dependent decrease in cell proliferation, invasion, migration, anchorage-independent growth and increased apoptosis in vitro. These results were recapitulated in vivo where oral administration of SAM reduced tumor volume and metastasis in green fluorescent protein (GFP)-tagged MDA-MB-231 xenograft model. Gene expression analyses validated the ability of SAM to decrease the expression of several key genes implicated in cancer progression and metastasis in both cell lines and breast tumor xenografts. SAM was found to be bioavailable in the serum of experimental animals as determined by enzyme-linked immunosorbent assay and no notable adverse side effects were seen including any change in animal behavior. The results of this study provide compelling evidence to evaluate the therapeutic potential of methylating agents like SAM in patients with breast cancer to reduce cancer-associated morbidity and mortality.

Identification of an Epigenetic Signature of Osteoporosis in Blood DNA of Postmenopausal Women.

Osteoporosis is one of the most common age-related progressive bone diseases in elderly people. Approximately one in three women and one in five men are predisposed to developing osteoporosis. In postmenopausal women, a reduction in BMD leads to an increased risk of fractures. In the current study, we delineated the DNA methylation signatures in whole blood samples of postmenopausal osteoporotic women. We obtained whole blood DNA from 22 normal women and 22 postmenopausal osteoporotic women (51 to 89 years old) from the Canadian Multicenter Osteoporosis Study (CaMos) cohort. These DNA samples were subjected to Illumina Infinium human methylation 450 K analysis. Illumina 450K raw data were analyzed by Genome Studio software. Analysis of the female participants with early and advanced osteoporosis resulted in the generation of a list of 1233 differentially methylated CpG sites when compared with age-matched normal women. T test, ANOVA, and post hoc statistical analyses were performed, and 77 significantly differentially methylated CpG sites were identified. From the 13 most significant genes, ZNF267, ABLIM2, RHOJ, CDKL5, and PDCD1 were selected for their potential role in bone biology. A weighted polygenic DNA methylation score of these genes predicted osteoporosis at an early stage with high sensitivity and specificity and correlated with measures of bone density. Pyrosequencing analysis of these genes was performed to validate the results obtained from Illumina 450 K methylation analysis. The current study provides proof of principal for the role of DNA methylation in osteoporosis. Using whole blood DNA methylation analysis, women at risk of developing osteoporosis can be identified before a diagnosis of osteoporosis is made using BMD as a screening method. Early diagnosis will help to select patients who might benefit from early therapeutic intervention. © 2018 American Society for Bone and Mineral Research.

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 alpha5beta1 and alphavbeta3 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.

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 x 10(5)) 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.

Prostate secretory protein PSP-94 decreases tumor growth and hypercalcemia of malignancy in a syngenic in vivo model of prostate cancer.

Prostate cancer is a common malignancy affecting men, which is often associated with skeletal metastases resulting in significant morbidity and mortality. In this hormone-dependent cancer, low levels of a prostate secretory protein of 94 amino acids (PSP-94) are associated with advanced disease stage. In the current study, we have examined the effect of PSP-94 on prostate cancer growth and experimental metastases to the skeleton. For these studies, MatLyLu rat prostate cancer cells were transfected with full-length cDNA encoding parathyroid hormone-related protein [PTHrP (MatLyLu-PTHrP cells)], which is known to be the major pathogenetic factor for malignancy-associated hypercalcemia. MatLyLu-PTHrP cells were inoculated s.c. into the right flank or via intracardiac route into the left ventricle of syngeneic male Copenhagen rats. Intracardiac inoculation of MatLyLu cells routinely results in the development of tumors in the lumbar vertebrae, resulting in hind-limb paralysis. Animals were infused with different doses of PSP-94 (0.1, 1.0, and 10.0 micro g/kg/day) starting on the day of tumor cell inoculation. Time of hind-limb paralysis and tumor volume were determined, and comparison was made between PSP-94-treated animals and control animals receiving vehicle alone. At the end of the study, animals were sacrificed, and plasma calcium, plasma PTHrP, and tumor PTHrP levels were determined. Whereas the highest dose of PSP-94 caused a modest but statistically significant delay in the development of hind-limb paralysis, a marked dose-dependent decrease in primary tumor volume was seen in experimental animals receiving PSP-94 due to its ability to promote tumor cell apoptosis. Furthermore, whereas control animals routinely developed hypercalcemia due to PTHrP production, treatment with PSP-94 led to a near normalization of plasma calcium and a marked reduction in PTHrP production as determined by radioimmunoassay and immunohistochemistry. Collectively, these results demonstrate the ability of PSP-94 to be an effective treatment modality for prostate cancer, where decrease in plasma PTHrP and calcium levels can serve as useful biochemical markers for monitoring the efficacy of this novel antitumor agent.

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 microg/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 microg/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.