Subhasree Nag

The study of pharmacokinetics (PK) and pharmacodynamics (PD) in cancer drug discovery and development is often paired and described in reciprocal terms, where PK is the analysis of the change in drug concentration with time and PD is the analysis of the biological effects of the drug at various concentrations over different time courses. While PK is defined by how a compound is absorbed, distributed, metabolized, and eliminated, PD refers to the measure of a compound’s ability to interact with its intended target, leading to a biologic effect. Recent advances in anti-breast cancer drug discovery have resulted in several new drugs, but there is still a high attrition rate during clinical development. One reason for this failure is attributed to inappropriate correlation between the PK and PD parameters and subsequent extrapolation to human subjects. In this chapter, we describe the protocols of PK and PD studies in breast cancer models to assess the efficacy of an anti-breast cancer compound, noting the types and endpoints employed, and explain why it is important to link PK and PD in order to establish and evaluate dose/concentration-response relationships and subsequently describe and predict the effect-time courses for a given drug dose.

RYBP (RING 1 and YY1-binding protein) is a newly identified member of the Polycomb group (PcG) proteins. PcG proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins are crucial for many biological processes, including self-renewal and differentiation, and cancer. Studies from our group and others suggest that RYBP has non-PcG functions and is downregulated in human cancers, including hepatocellular carcinoma (HCC). However, the potential role of RYBP in HCC is largely unknown. In the present study, for the first time, we systemically investigated the levels of RYBP expression and the linkage between RYBP deregulation and survivals of patients with HCC in 400 pairs of human HCC tissues and matched noncancerous samples. Using in vitro and in vivo HCC models, we determined the effort of RYBP on the survival of several HCC cell lines, including HepG2, Huh7 and Hep3B cells. We first verified that RYBP was downregulated in human HCC tumor specimens and cell lines, and that a low level of RYBP predicted a poor prognosis in HCC patients. We further demonstrated that enforced RYBP expression exerted potent effects against the various cell lines, leading to a significant decrease in cell viability, and inhibited cell colony formation and invasion, and induced apoptosis. We also found that the expression of numerous apoptosis- and metastasis-associated proteins was affected by RYBP. It reduced the expression of p53 in HepG2 cells, and increased Bax, cleaved-PARP and E-cadherin and, conversely, decreased Vimentin expression in all three HCC cells. Moreover, apoptosis was the main mechanism by which the RYBP exerted its cytostatic effects in HCC cells, while knockdown of RYBP showed opposite effects. In addition, we showed that AdRYBP infection inhibited the growth of tumors and induced RYBP expression in HepG2 and Huh7 xenograft models. There were no significant host toxicity and no gross organ abnormalities upon necropsy. We also determined that chemotherapeutic agents induced RYBP protein expression, and that RYBP sensitized HCC tumors to conventional chemotherapy through the induction of apoptosis both in vitro and in vivo. The results of these studies provide initial evidences supporting that the restoration of RYBP expression may be a new approach to targeted therapy for HCC and that RYBP may be a useful biomarker for predicting the prognosis of patients with HCC. (This work was supported by NIH/NCI grants R01 CA112029, R01 CA121211, and R01 CA186662 (to R.Z.); and NSFC 81272725 (to J.F.), NSFC 81372648 (to Q.G.) and Shanghai “Promising Youth Medical Worker” Project 13Y055 (to Q.G.) Citation Format: Wei Wang, Jianwen Cheng, Jiang-Jiang Qin, Sukesh Voruganti, Subhasree Nag, Jia Fan, Qiang Gao, Ruiwen Zhang. RYBP expression predicts survival of patients with hepatocellular carcinoma, and regulates response to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2015-5266

Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non‐classical‐Pc‐functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG‐independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the Pc‐repressive and non‐classical‐Pc‐functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy.

The activation of nuclear factor-kappaB (NFκB), a proinflammatory transcription factor, is a commonly observed phenomenon in breast cancer. It facilitates the development of a hormone-independent, invasive, high-grade, and late-stage tumor phenotype. Moreover, the commonly used cancer chemotherapy and radiotherapy approaches activate NFκB, leading to the development of invasive breast cancers that show resistance to chemotherapy, radiotherapy, and endocrine therapy. Inhibition of NFκB results in an increase in the sensitivity of cancer cells to the apoptotic effects of chemotherapeutic agents and radiation and restoring hormone sensitivity, which is correlated with increased disease-free survival in patients with breast cancer. In this review article, we focus on the role of the NFκB signaling pathways in the development and progression of breast cancer and the validity of NFκB as a potential target for breast cancer prevention and therapy. We also discuss the recent findings that NFκB may have tumor suppressing activity in certain cancer types. Finally, this review also covers the state-of-the-art development of NFκB inhibitors for cancer therapy and prevention, the challenges in targeting validation, and pharmacology and toxicology evaluations of these agents from the bench to the bedside.

RYBP is a member of the polycomb group (PcG) proteins that typically act as transcriptional repressors via epigenetic modification of chromatin. The present study was designed to investigate the role of RYBP in HCC progression, chemosensitivity, and patient survival, and to explore the underlying molecular mechanism(s). In this study we investigated the expression of RYBP in 400 pairs of human HCC tissues and matched noncancerous samples. The effects of RYBP on HCC tumor growth and metastasis and chemosensitivity were determined both in vitro and in vivo. We herein demonstrate that the RYBP expression in HCC tissue samples was significantly lower than that in matched noncancerous liver tissues. Clinically, the low expression of RYBP was an independent predictor of a poor prognosis in patients with HCC. In in vitro HCC models, enforced RYBP expression inhibited cell growth and invasion, induced apoptosis, and increased the chemosensitivity of the cells, while RYBP knockdown led to the opposite effects. Furthermore, RYBP expression was induced by cisplatin, and adenovirus-mediated RYBP expression inhibited HCC tumor growth and sensitized HCC to conventional chemotherapy in vivo. Our results demonstrate that reactivating RYBP in cancer cells may provide an effective and safe therapeutic approach to HCC therapy.

There is an increasing interest in targeting the MDM2 oncogene for cancer therapy. SP‐141, a novel designed small molecule MDM2 inhibitor, exerts excellent in vitro and in vivo anticancer activity. To facilitate the preclinical development of this candidate anticancer agent, we have developed an HPLC method for the quantitative analysis of SP‐141. The method was validated to be precise, accurate, and specific, with a linear range of 16.2–32,400 ng/mL in plasma, 16.2–6480 ng/mL in homogenates of brain, heart, liver, kidneys, lungs, muscle and tumor, and 32.4–6480 ng/mL in spleen homogenates. The lower limit of quantification was 16.2 ng/mL in plasma and all the tissue homogenates, except for spleen homogenates, where it was 32.4 ng/mL. The intra‐ and inter‐assay precisions (coefficient of variation) were between 0.86 and 13.39%, and accuracies (relative errors) ranged from −8.50 to 13.92%. The relative recoveries were 85.6–113.38%. SP‐141 was stable in mouse plasma, modestly plasma bound and metabolized by S9 microsomal enzymes. We performed an initial pharmacokinetic study in tumor‐bearing nude mice, demonstrating that SP‐141 has a short half‐life in plasma and wide tissue distribution. In summary, this HPLC method can be used in future preclinical and clinical investigations of SP‐141. Copyright © 2014 John Wiley & Sons, Ltd.

A requirement for Mouse Double Minute 2 (MDM2) oncogene activation has been suggested to be associated with cancer progression and metastasis, including breast cancer. To date, most MDM2 inhibitors have been designed to block the MDM2–p53-binding interphase, and have low or no efficacy against advanced breast cancer with mutant or deficient p53. Here we use a high-throughput screening and computer-aided, structure-based rational drug design, and identify a lead compound, SP-141, which can directly bind to MDM2, inhibit MDM2 expression and induce its autoubiquitination and proteasomal degradation. SP-141 has strong in vitro and in vivo antibreast cancer activity, with no apparent host toxicity. While further investigation is needed, our data indicate that SP-141 is a novel targeted therapeutic agent that may especially benefit patients with advanced disease.