Zhen Feng

Steroid Receptor Coactivator-1 Upregulates Integrin α5 Expression to Promote Breast Cancer Cell Adhesion and Migration

Metastatic breast cancer remains a lethal disease with poorly understood molecular mechanisms. Steroid receptor coactivator-1 (SRC-1 or NCOA1) is overexpressed in a subset of breast cancers with poor prognosis. It potentiates gene expression by serving as a coactivator for nuclear receptors and other transcription factors. We previously reported that SRC-1 promotes breast cancer metastasis without affecting primary mammary tumor formation. Herein, we found that SRC-1 deficiency in mouse and human breast cancer cells substantially reduced cell adhesion and migration capabilities on fibronectin and significantly extended the time of focal adhesion disassembly and reassembly. In agreement with this phenotype, SRC-1 expression positively correlated with integrin α(5) (ITGA5) expression in estrogen receptor-negative breast tumors whereas SRC-1 deficiency decreased ITGA5 expression. Furthermore, ITGA5 reduction in SRC-1-deficient/insufficient breast cancer cells or knockdown of ITGA5 in SRC-1-expressing breast cancer cells was associated with a disturbed integrin-mediated signaling. Critical downstream changes included reduced phosphorylation and/or dampened activation of focal adhesion kinase, paxillin, Rac1, and Erk1/2 during cell adhesion. Finally, we found that SRC-1 enhanced ITGA5 promoter activity through an AP-1 (activator protein)-binding site proximal to the transcriptional initiation site; both SRC-1 and c-Jun were recruited to this promoter region in breast cancer cells. These results show that SRC-1 can promote breast cancer metastasis by directly enhancing ITGA5 expression and thus promoting ITGA5-mediated cell adhesion and migration. Therefore, targeting ITGA5 in SRC-1-positive breast cancers may result in inhibition of SRC-1-promoted breast cancer metastasis.

NCOA1 Directly Targets M-CSF1 Expression to Promote Breast Cancer Metastasis

In breast cancer, overexpression of the nuclear coactivator NCOA1 (SRC-1) is associated with disease recurrence and resistance to endocrine therapy. To examine the impact of NCOA1 overexpression on morphogenesis and carcinogenesis in the mammary gland (MG), we generated MMTV-hNCOA1 transgenic [Tg(NCOA1)] mice. In the context of two distinct transgenic models of breast cancer, NCOA1 overexpression did not affect the morphology or tumor-forming capability of MG epithelial cells. However, NCOA1 overexpression increased the number of circulating breast cancer cells and the efficiency of lung metastasis. Mechanistic investigations showed that NCOA1 and c-Fos were recruited to a functional AP-1 site in the macrophage attractant CSF1 promoter, directly upregulating colony-simulating factor 1 (CSF1) expression to enhance macrophage recruitment and metastasis. Conversely, silencing NCOA1 reduced CSF1 expression and decreased macrophage recruitment and breast cancer cell metastasis. In a cohort of 453 human breast tumors, NCOA1 and CSF1 levels correlated positively with disease recurrence, higher tumor grade, and poor prognosis. Together, our results define an NCOA1/AP-1/CSF1 regulatory axis that promotes breast cancer metastasis, offering a novel therapeutic target for impeding this process.

Poly (γ, L-glutamic acid)-cisplatin bioconjugate exhibits potent antitumor activity with low toxicity: a comparative study with clinically used platinum derivatives.

We have recently synthesized a new platinum derivative, poly (γ, L‐glutamic acid)‐cisplatin conjugate (γ‐PGA‐CDDP), and shown that it displayed remarkable antitumor activity against breast tumor in a mouse model. The purpose of this study is to systematically compare this new drug with three platinum derivatives currently used in the clinic: cisplatin, carboplatin and oxaliplatin. Here, we show that γ‐PGA‐CDDP displays impressive antitumor activity over the current clinically used platinum drugs. More interestingly and more importantly, γ‐PGA‐CDDP conjugate significantly reduces cytotoxicity, mitigates oxidative stress and improves antioxidative capability in vivo. Animals treated with γ‐PGA‐CDDP display the same profile of body weight as the control animals, while the tumors in γ‐PGA‐CDDP‐treated animals are significantly suppressed compared with those treated with carboplatin and oxaliplatin. Our data suggest that γ‐PGA could be used as an effective carrier for drug delivery and that γ‐PGA‐CDDP conjugate may have potential therapeutic applications in human cancers that are sensitive to treatment with CDDP‐based chemotherapy such as ovarian cancer. (Cancer Sci 2010; 101: 2476–2482)

Addition of a cysteine to glucagon-like peptide-1 (GLP-1) conjugates GLP-1 to albumin in serum and prolongs GLP-1 action in vivo

Glucagon-like peptide-1 (GLP-1) is a promising new therapeutic agent for the treatment of type 2 diabetes. However, GLP-1 has a short half-life (t(1/)(2)<2min) due to rapid degradation by dipeptidyl peptidase IV in vivo. To circumvent this problem, a recombinant mGLP-1 with a cysteine at the C-terminus of GLP-1 was expressed in Escherichia coli and purified by affinity and reverse-phase chromatography. This addition of a cysteine facilitates mGLP-1 binding to serum albumin both in vitro and in vivo, thus protecting mGLP-1 from protease degradation. Similar to GLP-1, mGLP-1 stimulated cAMP production in PC12 cells and exhibited insulinotropic activity in MIN6 cells under in vitro culture conditions. Importantly, in glucose tolerance tests mice treated with mGLP-1 exhibited much lower glucose levels and much higher insulin levels versus that in mice treated with unmodified GLP-1. Furthermore, the effects of mGLP-1 on reduction of blood glucose levels lasted for 6-7h, while the effects of unmodified GLP-1 only lasted for 0.5-1h after injection. These results demonstrate that mGLP-1 is biologically active and its pharmaceutical efficacy is largely enhanced by the cysteine-mediated covalent conjugation with albumin in the serum after injection. Therefore, the mGLP-1 with a cysteine may be a better potential therapeutic drug than the unmodified GLP-1 for treating type 2 diabetes.

Effects of Insulin-Mimetic Vanadyl-Poly(γ-Glutamic Acid) Complex on Diabetic Rat Model

Poly-gamma-glutamic acid (gamma-PGA) prepared by fermentation of microbe was used as drug carrier for vanadium sulfate to obtain vanadyl-poly-gamma-glutamic acid (VO-gamma-PGA) complex. The FI-IR spectrum of the complex demonstrated that the expected VO-gamma-PGA complex is formed by the coordination of VO(2+) through the side chain carboxylic groups of the gamma-PGA. Studies of the complex in treating type I diabetes were carried out on alloxan induced diabetes rats. The results of treating the rats in 2 weeks and then stopping administration for 10 days showed that VO-gamma-PGA can effectively lower blood glucose levels of diabetic rats during administration. But after ceasing treatment there were no differences between groups in blood glucose level and water intake. The results of oral glucose tolerance and some serum parameters also demonstrated that VO-gamma-PGA was more effective than vanadium sulfate in treating diabetic rats.

Synthesis and characterization of cisplatin-loaded, EGFR-targeted biopolymer and in vitro evaluation for targeted delivery.

The design of smart targeted drug delivery systems that deliver drugs to specific cancer cells will give rise to cancer treatments with better efficacy and lower toxicity levels. We report the development and characterizations of maleimide-functionalized biopolymer (Mal-PGA-Asp) as an effective targeted drug delivery carrier synthesized from an amidation reaction between aspartylated PGA (PGA-Asp) and N-(maleimidohexanoyl)-ethylenediamine (NME). The epidermal growth factor receptor (EGFR) targeting peptide (TP13) was conjugated to Mal-PGA-Asp to obtain the targeting carrier (TP13-Mal-PGA-Asp). Cisplatin was finally loaded by complexation to form a biocompatible and tumor targeted therapeutic drug (TP13-Mal-PGA-Asp3-Pt). The resultant biopolymer with an average size 87 ± 28 nm showed a sustainable release profile with a half-maximal release time (t(1/2)) of approximately 15 h in physiological saline. Fluorescence imaging and flow cytometry analysis revealed that TP13 significantly enhanced the cellular uptake of TP13-Mal-PGA-Asp3-Pt in the human hepatoma cell line SMMC-7721. The IC(50) value demonstrated the superior anticancer activity of TP13-Mal-PGA-Asp3-Pt over PGA-Asp-Pt. Therefore, the newly developed drug carrier (TP13-Mal-PGA-Asp) obtained in this study may provide an efficient and targeted delivery of anticancer drugs, presenting a promising targeted chemotherapy in EGFR-positive cancers.

Breast tumor cell-specific knockout of Twist1 inhibits cancer cell plasticity, dissemination, and lung metastasis in mice

Significance We found that Twist1 is coexpressed with multiple other epithelial–mesenchymal transition (EMT)-inducing transcription factors (TFs) in small subsets of primary and circulating breast tumor cells. Together, they program a partial EMT and a basal-like phenotype of tumor cells, which most likely are responsible for disseminating into the circulation and metastasizing to the lung in mice. Targeting Twist1 largely diminishes all the cellular features and phenotypes of breast tumor cells, indicating Twist1 may be a target for inhibiting breast cancer metastasis. These findings are in favor of the role of partial EMT in breast cancer progression toward the metastatic state. Twist1 is an epithelial–mesenchymal transition (EMT)-inducing transcription factor (TF) that promotes cell migration and invasion. To determine the intrinsic role of Twist1 in EMT and breast cancer initiation, growth, and metastasis, we developed mouse models with an oncogene-induced mammary tumor containing wild-type (WT) Twist1 or tumor cell-specific Twist1 knockout (Twist1TKO). Twist1 knockout showed no effects on tumor initiation and growth. In both models with early-stage tumor cells, Twist1, and mesenchymal markers were not expressed, and lung metastasis was absent. Twist1 expression was detected in ∼6% of the advanced WT tumor cells. Most of these Twist1+ cells coexpressed several other EMT-inducing TFs (Snail, Slug, Zeb2), lost ERα and luminal marker K8, acquired basal cell markers (K5, p63), and exhibited a partial EMT plasticity (E-cadherin+/vimentin+). In advanced Twist1TKO tumor cells, Twist1 knockout largely diminished the expression of the aforementioned EMT-inducing TFs and basal and mesenchymal markers, but maintained the expression of the luminal markers. Circulating tumor cells (CTCs) were commonly detected in mice with advanced WT tumors, but not in mice with advanced Twist1TKO tumors. Nearly all WT CTCs coexpressed Twist1 with other EMT-inducing TFs and both epithelial and mesenchymal markers. Mice with advanced WT tumors developed extensive lung metastasis consisting of luminal tumor cells with silenced Twist1 and mesenchymal marker expression. Mice with advanced Twist1TKO tumors developed very little lung metastasis. Therefore, Twist1 is required for the expression of other EMT-inducing TFs in a small subset of tumor cells. Together, they induce partial EMT, basal-like tumor progression, intravasation, and metastasis.

A Novel GAP460 Biopolymer for Use as a Carrier in Drug-Delivery Applications

We synthesized a new non-toxic biopolymer (GAP460) containing γ,L-glutamic acid and aspartate (Asp). Conjugates of GAP460 and cisplatin exhibited a drug-carrying capacity of nearly 40%, 3-times higher than γ-PGA and dramatically decreasing the amount of biopolymer required for high-dose delivery. Treatment with GAP460-cisplatin conjugate (PACC) not only effectively inhibited tumor growth in nude mice, but also resulted in extended survival and lower nephrotoxicity, suggesting that GAP460 could be used as an effective carrier for drug delivery and that PACC may have potential therapeutic applications in the clinical treatment of cancer.

Abstract 2694: SRC-1 promotes tumor angiogenesis through up-regulation of HIF1α-mediated VEGFa expression.

Steroid receptor coactivator 1 (SRC-1) belongs to the p160 coactivator family which also contains SRC-2 (TIF2) and SRC-3 (AIB1/pCIP/ACTR/RAC3). These coactivators interact with nuclear receptors and other transcription factors to up-regulate gene expression. Accumulated evidence shows that SRC-1 exerts its promotive role in breast cancer progression and metastasis by up-regulating transcription of multiple genes important for tumor cell epithelial-mesenchymal transition (EMT), migration, invasion and endocrine therapy resistance. However, its role in tumor angiogenesis has not been examined. In our recent studies using SRC-1 KO/PyMT and WT/PyMT mammary tumor cell lines established in our lab, we found that injection of SRC-1 KO/PyMT cells to matrigel plug formed under the back skin in SCID mice induced less blood vessel formation compared with that of WT/PyMT tumor cells. Accordingly, injection of human breast cancer MDA-MB-231 cells with stable knockdown of SRC-1 by shRNA to the matrigel plug also induced less neovascularization. Meanwhile, VEGFa mRNA level is significantly reduced in mammary tumors from SRC-1 KO/PyMT transgenic mice. On the contrary, in SRC-1-overexpressed mammary tumors from MMTV/TVA/PyMT mice, the expression of VEGFa was dramatically increased. In agreement with these findings, in vitro studies revealed that VEGFa mRNA levels are much lower in two of the SRC-1 KO/PyMT tumor cell lines compared with WT/PyMT cell lines. Knockdown of SRC-1 in WT/PyMT cells and human MDA-MB-231 cells reduced VEGFa levels, while adenovirus-mediated re-expression of SRC-1 in SRC-1 KO/PyMT cells significantly potentiated VEGFa expression. Furthermore, promoter analyze revealed that SRC-1 could interact with HIF-1α and AP-1 to potentiate the VEGFa promoter activity. We also found that both SRC-1 and HIF-1α are associated with a VEGFa promoter region by ChIP assays. These results indicate that SRC-1 can promote breast cancer progression and metastasis through enhancing tumor angiogenesis. Targeting SRC-1 may be a feasible strategy for inhibiting breast tumor angiogenesis and metastasis. Citation Format: Li Qin, Yixiang Xu, Zhen Feng, Yelin Wu, Yan Xu, Lan Liao, Jianming Xu. SRC-1 promotes tumor angiogenesis through up-regulation of HIF1α-mediated VEGFa expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2013-2694 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1399: SRC-1 up-regulates CSF-1 expression to potentiate breast cancer metastasis

Steroid receptor coactivator-1 (SRC-1/NCOA1) has been reported to be overexpressed in a subset of human breast cancer (BC) and its overexpression correlates with Her2 expression, disease recurrence and endocrine therapy resistance. To determine the role and underlying mechanisms of SRC-1 overexpression in BC progression and metastasis, a transgenic mouse model with overexpression of human SRC-1 in mouse mammary epithelial cells was created. In vivo data revealed that overxpression of SRC-1 potentiated BC metastasis to lung in both MMTV/Neu and MMTV/TVA/PyMT tumor models. While SRC-1 overexpression did not obviously accelerate tumor growth in these BC mouse models, the number of tumor cells invaded into blood vessels and metastasis index in lung tissue significantly increased in these mice. In addition to the elevated Twist expression and activation of Her2 and AKT driven by SRC-1 overexpression in the tumors, CSF-1 level was significantly increased in SRC-1 overexpressed tumors. We also found that CSF-1 expression was decreased in two PyMT/SRC-1 KO tumor cell lines compared with the two PyMT/WT control cell lines. Knockdown of SRC-1 in PyMT/WT cells or human BC cells reduced CSF-1 levels, while adenoviral-mediated expression of SRC-1 in PyMT/SRC-1 KO cells or human BC cells significantly promoted CSF-1 expression. Furthermore SRC-1 was found to interact with transcription factors, c-jun and c-fos, to activate the csf-1 promoter. Both SRC-1 and c-fos were found to associate with the csf-1 promoter at AP-1 binding sites in human BC cells. This study provided compelling evidence that overexpression of SRC-1 promotes BC metastasis. Targeting SRC-1 might be a potential therapeutic strategy for controlling BC metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1399. doi:1538-7445.AM2012-1399