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PDF - 98K, Effect of PF-2771 on breast cell line proliferation and CIN index analysis.

PDF - 68K, Broad kinase selectivity analysis of PF-2771.

PDF - 76K, Cell cycle analysis of PF-2771 treatment of MDA-MB-468 tumor cells.

PDF - 165K, Comparison of the effect of PF-2771 and a Eg5/KSP inhibitor on the microtubule network.

PDF - 238K, Supplementary Materials and Methods

PDF - 219K, PK/PD/Efficacy model analysis.

Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E–dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic–pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer. Mol Cancer Ther; 13(8); 2104–15. ©2014 AACR.

PDf - 114K, Analysis of mouse body weight in the tumor growth models.

Nutraceutical is a food, or part of a food, used for the prevention and/or treatment of diseases. A number of nutraceuticals serve as candidates for development of prostate cancer chemopreventive agents because of promising epidemiological, preclinical and pilot clinical findings. Their mechanisms of action may involve an ability to target multiple molecular pathways in carcinogenesis without eliciting toxic side effects.This review provides an overview of several nutraceuticals, including green tea polyphenol, omega-3 fatty acids, vitamin D, lycopene, genistein, quercetin, resveratrol and sulforaphane, for the clinical relevance to chemoprevention of prostate cancer. Their mechanisms of action on regulating key processes of carcinogenesis are also discussed. For each of these agents, a brief summary of completed or currently ongoing clinical trials related to the chemopreventive efficacy on prostate cancer is given.Even though a few clinical trials have been conducted, review of these results indicate that further studies are required to confirm the clinical efficacy and safety, and to provide a guidance on how to use nutraceuticals for optimal effect. Future cancer prevention clinical trials for the nutraceuticals should recruit men with an increased risk of prostate cancer.

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K i

The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and their biological activity is mediated by the formation of heterodimeric complexes of BMP type I and type II receptors of serine/threonine kinases. BMPs play an important role in regulating cell proliferation, differentiation and migration, processes that are involved in carcinogenesis of many types of cancer. BMP-2 has been shown overexpressed in human non-small cell lung cancer (NSCLC) and enhances lung tumorigenesis, suggesting that blocking the BMP signaling may be an effective therapeutic approach for NSCLC. Although previous work demonstrated that known protein-based BMP antagonists such as Noggin may reduce lung tumor growth, their clinical application could be limited by short half-lives and poor intra-tumor delivery. In a previous zebrafish embryo-based structure and activity study, we identified DMH1, a highly selective small molecule inhibitor which specifically antagonize the intracellular kinase domains of BMP type I receptors, the critical mediators of BMP signaling. In the present study, we found that DMH1 potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in vitro by blocking the BMP signaling, as indicated by the reduction of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3 in A549 and H460 cells. In an in vivo study, treatment by intraperitoneal injection of DMH1 (5 mg/kg) for four weeks significantly reduced the growth of A549-derived xenograft in SCID mice. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel therapeutic strategy for lung cancer. Citation Format: Ying Huang, Rachel Lee, Andy Chang, Jeffery Fan, Chantelle Labib, Cyrus Parsa, Robert Orlando, Bradley Andresen, Jijun Hao. DMH1, a small molecule inhibitor of BMP type I receptors, suppresses growth and invasion of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2717. doi:10.1158/1538-7445.AM2014-2717

Background Si-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. In our previous study, the microarray gene expression profiles of SWT on breast cancer cell line MCF-7 were found similar to the effect of β-estradiol (E2) on MCF-7 cells in the Connectivity Map database. Methods Further data analysis was conducted to find the main similarities and differences between the effects of SWT and E2 on MCF-7 gene expression. The cell proliferation assay on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) cells were used to examine such estrogenic activity. The estrogenic potency of SWT was further confirmed by estrogen-responsive element (ERE) luciferase reporter assay in MCF-7 cells. Results Many estrogen regulated genes strongly up-regulated by E2 were similarly up-regulated by SWT, e.g., GREB1, PGR and EGR3. Of interest with regard to safety of SWT, the oncogenes MYBL1 and RET were strongly induced by E2 but not by SWT. Quantitative RT-PCR analysis revealed a highly concordant expression change in selected genes with data obtained by microarrays. Further supporting SWT’s estrogenic activity, in MCF-7 but not in MDA-MB-231 cells, SWT stimulated cell growth at lower concentrations (< 3.0 mg/ml), while at high concentrations, it inhibits the growth of both cell lines. The growth inhibitory potency of SWT was significantly higher in MDA-MB-231 than in MCF-7 cells. The SWT-induced cell growth of MCF-7 could be blocked by addition of the estrogen receptor antagonist tamoxifen. In addition, SWT was able to activate the ERE activity at lower concentrations. The herbal components Angelicae, Chuanxiong and Rehmanniae at lower concentrations (< 3.0 mg/ml) also showed growth-inducing and ERE-activating activity in MCF-7 cells. Conclusions These results revealed a new mechanism to support the clinical use of SWT for estrogen related diseases and possibly for cancer prevention. This study also demonstrated the feasibility of using microarray transcriptional profiling to discover phytoestrogenic components that are present in natural products.

Chemoprevention is one of the strategies for cancer control. Chinese herbal medicines provide a promising source toward developing low cost and low toxic chemopreventive agents. Si-Wu-Tang (SWT) is a traditional Chinese medicine that has been used in Asian countries for about 1,000 years for women's health. SWT contains four herbs: Rehmanniae, Angelica, Chuanxiong and Paeoniae. It also contains at least nine detectable compounds (Compounds 1 – 9). From our previous study, we found that in human breast cancer MCF-7 cells treated with SWT, the genes in the Nrf2 cytoprotective pathways were strongly induced (Wen et al, PLoS ONE, 2011), suggesting that SWT may have antioxidant and chemopreventive activity. To evaluate such activity, we used murine epidermal cell JB6-based soft agar transformation assay, which is a model extensively used to study tumor promotion and chemopreventive activity. JB6 cells were seeded in 96-well plates in 0.33% agar suspended on top of a layer of 0.5% agar. EGF (10 ng/mL) was used to promote the anchorage-independent growth of JB6 cells on soft agar, which is one of the hallmarks of cell transformation. Various concentrations of SWT, components and compounds were added together with EGF into the top and bottom layers of the agar. Colonies were counted 7 to 10 days after the treatment. SWT showed a dose-dependent inhibition of colony formation at non-toxic concentrations: 0.0256, 0.256 and 1.28 mg/mL inhibited 56.5, 79.6 and 93.2 % colony formation compared to the EGF only control, respectively. Each of the four individual herbal components of SWT showed certain degrees of inhibitory effects on colony formation at a high concentration (2.56 mg/mL). The most potent component is Paeoniae, which at 0.0256 and 0.256 mg/mL inhibited 58.5 and 85.3 % of colony formation, respectively. Five pure compound constitutes of SWT were also tested in the soft agar assay. Compound 3 at 2.0 and 10 ug/mL inhibited 73.91% and 99.33% of colony formation, respectively. Compound 5 at 4.0 and 10 ug/mL inhibited 83.95 and 96.99% of colony formation, respectively. Compound 8 at 0.2 and 4.0 ug/mL inhibited 93.98% and 99.67% of colony formation, respectively. The concentration range tested did not inhibit cell proliferation as measured by Sulforhodamine B assay, therefore the inhibition of cell transformation by these compounds was not caused by growth inhibition or the effects of cytotoxicity. Other compounds tested failed to show any effect on colony formation. In conclusion, this study demonstrates that SWT and its components were able to inhibit EGF-induced neoplastic transformation at non-toxic concentrations. This widely used Chinese medicinal formula may provide a promising option toward preventing many types of cancer. Citation Format: Mandy Mei Liu, Jeffery Fan, Zhijun Wang, Zhong Zuo, Moses Chow, Ying Huang. Evaluation of cancer preventive activity of Chinese medicinal formula Si-Wu-Tang. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B50.