Jin-Qiu Chen

Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.

Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities.

Label-retaining liver cancer cells are relatively resistant to sorafenib.

Objective The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib. Methods We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib. Results LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. Conclusions Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.

The synthetic bryostatin analog Merle 23 dissects distinct mechanisms of bryostatin activity in the LNCaP human prostate cancer cell line

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.

Overlapping Substrate and Inhibitor Specificity of Human and Murine ABCG2

ABCG2 (also known as breast cancer resistance protein) is an ATP-binding cassette (ABC) transporter localized to the plasma membrane where it mediates the efflux of xenobiotics, including potential therapeutics. Studies investigating Abcg2 function at the blood-brain barrier in mouse models are often compared with human ABCG2 function. It is critical to understand the nature of species differences between mouse and human ABCG2, since extrapolations are made from murine data to humans. Two independent drug-selected cell line pairs expressing human or mouse ABCG2 were compared for efflux of fluorescent substrates using flow cytometry. To this end, we developed and characterized a new mouse Abcg2-expressing subline that demonstrated efflux of known fluorescent ABCG2 substrates and increased resistance to mitoxantrone, which is reduced in the presence of the ABCG2 inhibitor Ko143. Our results indicate that the substrate specificity of human and mouse ABCG2 is very similar. We identified a new human and mouse ABCG2 substrate, a porphyrin analog, purpurin-18 (Pp-18), which is not a substrate for P-glycoprotein or multidrug resistance protein 1. The ability of inhibitors to block efflux activity of ABCG2 was assessed using Pp-18. Inhibitors also demonstrated similar effects on human and mouse ABCG2. Chrysin, benzoflavone, and cyclosporin A inhibited Pp-18 efflux in both human and mouse ABCG2. The similarity of the substrate and inhibitor specificity of human and mouse ABCG2 supports interpretation of mouse models in understanding the clinical, pharmacological, and physiologic roles of ABCG2.

Pharmacodynamic markers and clinical results from the phase 2 study of the SMAC mimetic birinapant in women with relapsed platinum-resistant or -refractory epithelial ovarian cancer

Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. It was hypothesized that blocking IAPs with birinapant would increase tumor cell death and result in objective responses for women with platinum‐refractory and ‐resistant ovarian cancer.

Capillary isoelectric-focusing immunoassays to study dynamic oncoprotein phosphorylation and drug response to targeted therapies in non-small cell lung cancer

Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non–small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEK inhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal–regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 μg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib and MEK1 inhibitor treatments in an NSCLC patient. NanoPros higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPros investigation for future drug development and evaluation of drug effects of targeted therapies. Mol Cancer Ther; 12(11); 2601–13. ©2013 AACR.

Biological Profile of the Less Lipophilic and Synthetically More Accessible Bryostatin 7 Closely Resembles That of Bryostatin 1

The bryostatins are a group of 20 macrolides isolated by Pettit and co-workers from the marine organism Bugula neritina. Bryostatin 1, the flagship member of the family, has been the subject of intense chemical and biological investigations due to its remarkably diverse biological activities, including promising indications as therapy for cancer, Alzheimers disease, and HIV. Other bryostatins, however, have attracted far less attention, most probably due to their relatively low natural abundance and associated scarcity of supply. Among all macrolides in this family, bryostatin 7 is biologically the most potent protein kinase C (PKC) ligand (in terms of binding affinity) and also the first bryostatin to be synthesized in the laboratory. Nonetheless, almost no biological studies have been carried out on this agent. We describe herein the total synthesis of bryostatin 7 based on our pyran annulation technology, which allows for the first detailed biological characterizations of bryostatin 7 with side-by-side comparisons to bryostatin 1. The results suggest that the more easily synthesized and less lipophilic bryostatin 7 may be an effective surrogate for bryostatin 1.

Biological responses to TGF-β in the mammary epithelium show a complex dependency on Smad3 gene dosage with important implications for tumor progression.

TGF-β plays a dual role in epithelial carcinogenesis with the potential to either suppress or promote tumor progression. We found that levels of Smad3 mRNA, a critical mediator of TGF-β signaling, are reduced by approximately 60% in human breast cancer. We therefore used conditionally immortalized mammary epithelial cells (IMEC) of differing Smad3 genotypes to quantitatively address the Smad3 requirement for different biologic responses to TGF-β. We found that a two-fold reduction in Smad3 gene dosage led to complex effects on TGF-β responses; the growth-inhibitory response was retained, the pro-apoptotic response was lost, the migratory response was reduced, and the invasion response was enhanced. Loss of the pro-apoptotic response in the Smad3+/− IMECs correlated with loss of Smad3 binding to the Bcl-2 locus, whereas retention of the growth-inhibitory response in Smad3 IMECs correlated with retention of Smad3 binding to the c-Myc locus. Addressing the integrated outcome of these changes in vivo, we showed that reduced Smad3 levels enhanced metastasis in two independent models of metastatic breast cancer. Our results suggest that different biologic responses to TGF-β in the mammary epithelium are differentially affected by Smad3 dosage and that a mere two-fold reduction in Smad3 is sufficient to promote metastasis. Mol Cancer Res; 10(10); 1389–99. ©2012 AACR.

A Phase II Trial of AZD6244 (Selumetinib, ARRY-142886), an Oral MEK1/2 Inhibitor, in Relapsed/Refractory Multiple Myeloma.

Purpose: AZD6244 is a MEK1/2 inhibitor with significant preclinical activity in multiple myeloma cells. This phase II study used a two-stage Simon design to determine the AZD6244 response rate in patients with relapsed or refractory multiple myeloma. Experimental Design: AZD6244 (75 mg) was administered orally, twice a day, continuously for 28-day cycles. Response was evaluated after three cycles. Results: Thirty-six patients received therapy. The median age was 65 years (range: 43–81) and the median number of prior therapies was 5 (range: 2–11). The most common grade 3 and 4 toxicities included anemia, neutropenia, thrombocytopenia, diarrhea, and fatigue. Three deaths occurred possibly related to AZD6244 (2 due to sepsis, 1 due to acute kidney injury). After AZD6244 discontinuation, three additional deaths occurred due to disease progression. The response rate (CR + PR) was 5.6% with a mean duration of response of 4.95 months and median progression-free survival time of 3.52 months. One patient had a very good partial response (VGPR), 1 patient had a partial response, 17 patients had stable disease, 13 patients had progressive disease, and 4 patients could not be assessed for response. Pharmacodynamic studies revealed variable effects on bone marrow CD138+ cell MEK1/2 and ERK1/2 phosphorylation. The best clinical response, a prolonged VGPR, occurred in a patient with an MMSET translocation. Conclusions: Single-agent AZD6244 was tolerable and had minimal activity in this heavily pretreated population. Clin Cancer Res; 22(5); 1067–75. ©2015 AACR.

Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared with single agents. In addition, a breast cancer patient–derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared with single agents. Mice bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared with single agents. A set of 37 genes cooperatively affected (34 downregulated; 3 upregulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes downregulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared with normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC-binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo. Mol Cancer Ther; 16(9); 2008–21. ©2017 AACR.