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1.  Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases 
Oncogene  2013;33(8):986-995.
Interrogating specific cellular activities often entails the dissection of posttranslational modifications or functional redundancy conferred by protein families, which demands more sophisticated research tools than simply eliminating a specific gene product by gene targeting or RNAi. We have developed a novel methodology that involves engineering a single SCFβTrCP-based ubiquitin ligase that is capable of not only simultaneously targeting the entire family of ErbB receptor tyrosine kinases for ubiquitination and degradation, but also selectively recruiting only activated ErbBs. The engineered SCFβTrCP ubiquitin ligase effectively blocked ErbB signaling and attenuated oncogenicity in breast cancer cells, yet had little effect on the survival and growth of non-cancerous breast epithelial cells. Therefore, engineering ubiquitin ligases offers a simple research tool to dissect the specific traits of tumorigenic protein families, and provides a rapid and feasible means to expand the dimensionality of drug discovery by assessing protein families or posttranslational modifications as potential drug targets.
doi:10.1038/onc.2013.33
PMCID: PMC3930622  PMID: 23416973
2.  Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-κB 
Scientific Reports  2014;4:5750.
Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression.
doi:10.1038/srep05750
PMCID: PMC4102923  PMID: 25034888
3.  CCL2 mediates crosstalk between cancer cells and stromal fibroblasts that regulates breast cancer stem cells 
Cancer Research  2012;72(11):2768-2779.
Cancer stem cells (CSCs) play critical roles in cancer initiation, progression, and therapeutic refractoriness. Although many studies have focused on the genes and pathways involved in stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer (BC) stem cells. We found that compared to normal fibroblasts, primary cancer-associated fibroblasts (CAFs) and fibroblasts activated by co-cultured BC cells produce higher levels of chemokine (C-C motif) ligand 2 (CCL2), which stimulates the stem cell-specific, sphere-forming phenotype in BC cells and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3 activation by diverse BC-secreted cytokines, and in turn, induced NOTCH1 expression and the CSC features in BC cells, constituting a “cancer-stroma-cancer” signaling circuit. In a xenograft model of paired fibroblasts and BC tumor cells, loss of CCL2 significantly inhibited tumorigenesis and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated with poor differentiation in primary BCs, further supporting the observation that NOTCH1 is regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic target that can block the cancer-host communication that prompts CSC-mediated disease progression.
doi:10.1158/0008-5472.CAN-11-3567
PMCID: PMC3367125  PMID: 22472119
breast cancer; cancer stem cells; chemokine (C-C motif) ligand 2; tumor microenvironment; stromal fibroblasts
4.  Dysfunctional Activation of Neurotensin/IL-8 Pathway in Hepatocellular Carcinoma Is Associated with Increased Inflammatory Response in Microenvironment, More Epithelial Mesenchymal Transition in Cancer and Worse Prognosis in Patients 
PLoS ONE  2013;8(2):e56069.
Aim
To investigate the role of neurotensin (NTS) in hepatocellular carcinoma (HCC) sub- grouping and the clinical and pathological significance of activation of NTS/IL-8 pathway in HCC.
Methods
The genome-wide gene expression profiling were conducted in 10 pairs of cancer tissues and corresponding normal adjacent tissues samples using Affymetrix GeneChip® Human Genome U133 Plus 2.0 microarray to screen differentially expressing genes and enrich dysfunctional activated pathways among different HCC subgroups. The levels of NTS protein and multiple inflammation and epithelial mesenchymal transition (EMT) related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin, β-Catenin and Vimentin were examined in 64 cases of paraffin-embedded HCC samples using immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) were compared.
Results
A subgroup of HCC characterized by up-regulated NTS expression was accompanied by up-regulated inflammatory responses and EMT. The direct interaction between NTS and IL-8 was identified by pathway enrichment analysis. Significantly increased IL-8 protein was confirmed in 90.91% of NTS+ HCC samples and significantly positively correlated to the levels of NTS protein in cancer tissues (P = 0.036), which implied activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 correlated with co-expression of NTS and IL-8. Increased infiltration of CD68+ macrophages and more cancer cells displaying EMT features were found in NTS+IL-8+ samples. The co-expression of NTS and IL-8 in cancer significantly correlated with the clinical outcomes, as the mortality rate of NTS+IL-8+ HCC patients is 2.5-fold higher than the others after the surgery (P = 0.022). Accordingly, the OS of NTS+IL-8+ HCC patients significantly decreased who are under a higher hazard of death at an expected hazard ratio (HR) of 3.457.
Conclusion
Dysfunctional activation of the NTS/IL-8 pathway was detected in HCC which is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.
doi:10.1371/journal.pone.0056069
PMCID: PMC3572009  PMID: 23418512
5.  Combining the negative lymph nodes count with the ratio of positive and removed lymph nodes can better predict the postoperative survival in cervical cancer patients 
Background
To evaluate the impacts of the negative lymph nodes (NLNs) count on the prognostic prediction of the ratio of positive and removed lymph nodes (RPL) in cervical cancer patients after radical hysterectomy and pelvic lymphadenectomy (RHPL).
Methods
The positive and negative lymph node counts were calculated for 609 postoperative cervical cancer patients. The 5-year survival rate (5-YSR) was examined according to clinicopathologic variables. Cox regression was used to identify independent prognostic factors.
Results
The NLNs count cutoffs were determined to be 10 and 25 with 5-YSR of 62.8% and 80.5%. The RPL of 13 patients who had the NLNs count of 10 or fewer was >20%. Among 242 patients who had 10 < NLNs count ≤ 25, 194 without positive nodes had the 5-YSR of 77.8%, 31 with 0% < RPL ≤ 5% had the 5-YSR of 3.2%, 15 with RPL > 20% had died when follow-up was completed. Among 354 patients who had NLNs count >25, 185 without positive nodes had the 5-YSR of 87.6%, 6 with 0% < RPL ≤ 5% had the 5-YSR of 25%, 15 with 5% < RPL ≤ 20% had the 5-YSR of 4.5%, and 2 with RPL >20% had died when follow-up was completed. Furthermore, stage, histologic grade and RPL were independently correlated with overall survival of cervical cancer patients after RHPL in the multivariate analysis.
Conclusions
RPL was an independent prognostic factor. The NLNs count is a key factor for improvement of survival prediction of RPL in cervical cancer.
doi:10.1186/1475-2867-13-6
PMCID: PMC3576300  PMID: 23374254
Cervical cancer; Lymph node; Pelvic lymphadenectomy; Prognosis
6.  Nuclear Translocation of Type I Transforming Growth Factor β Receptor Confers a Novel Function in RNA Processing 
Molecular and Cellular Biology  2012;32(12):2183-2195.
Signaling of transforming growth factor β (TGF-β) is redirected in cancer to promote malignancy, but how TGF-β function is altered in a transformed cell is not fully understood. We investigated TGF-β signaling by profiling proteins that differentially bound to type I TGF-β receptor (TβRI) in nontransformed, HER2-transformed, and HER2-negative breast cancer cells using immunoprecipitation followed by protein identification. Interestingly, several nuclear proteins implicated in posttranscriptional RNA processing were uniquely identified in the TβRI coprecipitates from HER2-transformed cells. Ligand-inducible nuclear translocation of TβRI was observed only in transformed cells, and the translocation required importin β1, nucleolin, and Smad2/3. This trafficking was dependent on the high Ran GTPase activity resulting from oncogenic transformation. In the nucleus, TβRI associated with purine-rich RNA sequences in a synergistic manner with the RNA-binding factor hnRNP A1. We further found that nuclear translocation of TβRI specifically induced epidermal growth factor receptor (EGFR) transcript isoform c, which encodes a soluble EGFR protein, through alternative splicing or 3′-end processing. Our study confirms a cancer-specific nuclear translocation of TβRI and demonstrates its potential function in regulating nuclear RNA processing, as well as a novel gain-of-function mechanism of TGF-β signaling in cancer.
doi:10.1128/MCB.00320-12
PMCID: PMC3372271  PMID: 22473997
7.  Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor 
PLoS ONE  2012;7(5):e36418.
Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apcmin mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth.
doi:10.1371/journal.pone.0036418
PMCID: PMC3344856  PMID: 22574158
8.  De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer 
Background
MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.
Methods
The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.
Results
More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.
Conclusions
Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.
doi:10.1186/1479-5876-10-42
PMCID: PMC3342150  PMID: 22400902
Breast cancer; miRNA; Biomarker; Neoadjuvant chemotherapy; Metastasis
9.  Over-Expression of Semaphorin4D, Hypoxia-Inducible Factor-1α and Vascular Endothelial Growth Factor Is Related to Poor Prognosis in Ovarian Epithelial Cancer 
Semaphorin4D (SEMA4D) has been regarded as an important protein in tumor angiogenesis, though originally identified in neurodevelopment. SEMA4D is extensively expressed in several malignant solid tumors. Nevertheless, the function and expression of SEMA4D in epithelial ovarian cancer (EOC) is as yet not well understood. The aim of this study was to investigate SEMA4D expression in EOC and evaluate its clinical–pathological and prognostic significance. Immunohistochemistry was used to analyze SEMA4D expression and tumor angiogenesis-related proteins (HIF-1α and VEGF) in tissues from 40 patients with normal ovarian epithelia and 124 EOC patients. SEMA4D was found to be expressed in 61.3% of the 124 EOC tissues, which was significantly higher than in the normal ovarian epithelia (p < 0.001). SEMA4D expression correlated with HIF-1α and VEGF closely (ρ = 0.349 and 0.263, p < 0.001). Positive SEMA4D staining was significantly higher in tissues from patients with low histological grade, FIGO stage III-IV, lymph node metastasis and residual disease ≥1 cm (p < 0.05). In the Cox proportional hazard mode, SEMA4D expression and histologic grade were independent indicators of overall survival (OS) and progress-free survival (PFS) for EOC patients. These findings suggest that the cooperation of SEMA4D, HIF-1α, and VEGF may indicate poor prognosis for patients with EOC, thereby demonstrating that SEMA4D and its role in angiogenesis in EOC warrants further study.
doi:10.3390/ijms131013264
PMCID: PMC3497325  PMID: 23202951
Semaphorin4D; HIF-1; VEGF; ovarian cancer; prognosis
10.  Context-dependent bidirectional regulation of the mutS homolog 2 by transforming growth factor β contributes to chemoresistance in breast cancer cells 
Molecular cancer research : MCR  2010;8(12):1633-1642.
The transforming growth factor β (TGF-β), a tumor suppressive cytokine in normal cells, is abused in cancer to promote the malignancy. In this study we reported that TGF-β downregulated the mutS homolog 2 (MSH2), a central component of the DNA mismatch repair (MMR) system, in HER2-transformed MCF10A mammary epithelial cells and in breast cancer (BC) cells. This was mediated by a TGF-β-induced microRNA (miRNA), miR-21, which targeted the 3’ untranslated region (UTR) of MSH2 mRNA and downregulated its expression. A negative correlation between the expression of TGF-β1 and MSH2 was also detected in primary breast tumors. In contrast, TGF-β upregulated MSH2 in non-transformed cells through Smad-mediated, p53-dependent promoter activation, which was absent in BC cells with impaired p53 function. Although this upregulating mechanism also existed in MCF10A/HER2 and p53-proficient BC cells, both basal and TGF-β-induced MSH2 promoter activities were significantly lower than those in MCF10A. Moreover, the basal and TGF-β-induced miR-21 levels were markedly higher in transformed cells, suggesting that the pre-set levels of miR-21 and MSH2 promoter activity, which is affected by the p53 status, determine the outputs of the bidirectional regulation of MSH2 by TGF-β in a certain cellular context. We further found that by downregulating MSH2, TGF-β contributed to resistance to DNA-damaging chemotherapy agents in cancer cells. Our results indicated a regulatory antagonism between promoter activation and miRNA-mediated post-transcriptional inhibition underlying a dual effect of TGF-β on the DNA repair machinery, which may influence the genomic stability in a context-dependent manner and contribute to chemoresistance in cancer.
doi:10.1158/1541-7786.MCR-10-0362
PMCID: PMC3059495  PMID: 21047769
TGF-β; MSH2; DNA mismatch repair; breast cancer; chemoresistance
11.  Upregulated Expression of Indoleamine 2, 3-Dioxygenase in Primary Breast Cancer Correlates with Increase of Infiltrated Regulatory T Cells In Situ and Lymph Node Metastasis 
IDO has been reported to induce immunotolerance and promote metastasis in solid malignancy, but the mechanisms involved were not fully understood. In this study, the expression of IDO in primary breast cancer was examined and the correlation between the expression levels of IDO and the densities of Foxp3+ Tregs in situ was studied. The IDO stably-expressing CHO cells(IDO/CHO) were generated to evaluate the induction of Foxp3+ Tregs after coculturing with CD3+ T cells in vitro. The IDO expression in cancer was higher than that in benign diseases both at RNA and protein levels. The IDO expression was significantly upregulated in tumors of more advanced stages and with more extensive lymph node metastasis, and displayed positive linear correlation with the density of Foxp3+ Tregs. We further demonstrated that CD4+CD25+CD127− Tregs could be amplified by coculturing CD3+ T cells with IDO/CHO cells in vitro which displayed increasing Foxp3 expression both at mRNA and protein levels. Our results implied that up-regulation of IDO in primary breast cancer may inhibit local immune surveillance and promote metastasis by favoring development and infiltration of Foxp3+ Tregs in the tumor microenvironment.
doi:10.1155/2011/469135
PMCID: PMC3202140  PMID: 22110525
12.  Transforming growth factor β regulates the sphere-initiating stem cell-like feature in breast cancer through miRNA-181 and ATM 
Oncogene  2010;30(12):1470-1480.
Recent studies indicate that a subset of cancer cells possessing stem cell properties, referred to as cancer-initiating or cancer stem cells (CSCs), play crucial roles in tumor initiation, metastasis and resistance to anticancer therapies. Transforming growth factor (TGF)-βs and their family members have been implicated in both normal (embryonic and somatic) stem cells and CSCs. In this study, we observed that exposure to TGF-β increased the population of breast cancer (BC) cells that can form mammospheres in suspension, a feature endowed by stem cells. This was mediated by the micro(mi)RNA family miR-181, which was upregulated by TGF-β at the post-transcriptional level. Levels of the miR-181 family members were elevated in mammospheres grown in undifferentiating conditions, compared to cells grown in two dimensional (2D) conditions. Ataxia telangiectasia mutated (ATM), a target gene of miR-181, exhibited reduced expression in mammospheres and upon TGF-β treatment. Overexpression of miR-181a/b, or depletion of ATM or its substrate CHK2, was sufficient to induce sphere formation in BC cells. Finally, knockdown of ATM enhanced in vivo tumorigenesis of the MDA361 BC cells. Our results elucidate a novel mechanism through which the TGF-β pathway regulates the CSC property by interfering with the tumor suppressor ATM, providing insights into the cellular and environmental factors regulating CSCs, which may guide future studies on therapeutic strategies targeting these cells.
doi:10.1038/onc.2010.531
PMCID: PMC3063856  PMID: 21102523
TGF-β; cancer stem cells; microRNA; ATM; CHK2
13.  Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells 
Introduction
The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs) from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs.
Methods
An IDO expression plasmid was constructed and Chinese hamster ovary (CHO) cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control) CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127-]) subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression.
Results
IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127- T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P < 0.05) as well. The specific inhibitor of IDO,1-MT efficiently reversed enhancement of T cells apoptosis and amplification of Tregs in vitro. Increased expression of Foxp3, a key molecular marker of Tregs, was confirmed by RT-PCR, real-time RT-PCR and Western blot analysis at the same time.
Conclusions
These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.
doi:10.1186/1756-9966-30-82
PMCID: PMC3184069  PMID: 21917155
Indoleamine-Pyrrole 2; 3-Dioxygenase; breast neoplasms; immune tolerance; CHO Cells; regulatory T-Lymphocytes
14.  Tie2 signaling regulates osteoclastogenesis and osteolytic bone invasion of breast cancer 
Cancer research  2010;70(7):2819-2828.
Breast to bone metastasis is a common occurrence in the majority of patients with advanced breast cancer. The metastases are often incurable, are associated with bone destruction and high rates of morbidity. Understanding the underlying mechanisms of how metastatic tumor cells induce bone destruction is critically important. We previously reported that Tie2, a receptor tyrosine kinase, is significantly increased in human breast cancer tissues compared to normal and benign breast tumors, and regulates tumor angiogenesis. In this study, we identify a new function of Tie2 in osteoclastogenesis and osteolytic bone invasion of breast cancer. Tie2 is present in hematopoietic stem/precursor cells. Genetic deletion of Tie2 or neutralization of Tie2 function using soluble Tie2 receptor impaired osteoclastogenesis in an embryonic stem (ES) cell differentiation assay. In contrast, deletion of Tie2 has no effect on osteoblastogenesis. As CD11b myeloid cells have the potential to become osteoclast and Tie2 is present in certain population of these cells, we isolated Tie2+ and Tie2− myeloid cells. We observed a significant reduction of osteoclastogenesis in Tie2− compared to Tie2+ CD11b cells. Consistently, neutralization of Tie2 activity in vivo significantly inhibited osteolytic bone invasion and tumor growth in a mammary tumor model, which correlated with a significant reduction of osteoclast and tumor angiogenesis. Collectively, these data reveal a direct and novel role of Tie2 signaling in osteoclast differentiation. These findings identify Tie2 a therapeutic target for controlling not only tumor angiogenesis, but also osteolytic bone metastasis in breast cancer.
doi:10.1158/0008-5472.CAN-09-1915
PMCID: PMC2848896  PMID: 20233869
Breast cancer; bone metastasis; Tie2; osteoclast; osteoclastogenesis
15.  Oncogenic Mutations Regulate Tumor Microenvironment through Induction of Growth Factors and Angiogenic Mediators 
Oncogene  2010;29(23):3335-3348.
Activating mutations in the tyrosine kinase domain of HER2 (ErbB2) have been identified in human cancers. Compared to wild-type HER2, mutant HER2 shows constitutively activate kinase activity and increased oncogenicity. Cells transformed by mutant HER2 are resistant to EGFR tyrosine kinase inhibitors and exhibit an attenuated response to the HER2 antibody trastuzumab. We investigated herein pathways through which mutant HER2 alters the extracellular environment, potentially leading to drug resistance and the effect of simultaneously targeting HER2 and the tumor cell microenvironment with a therapeutic intent. Expression of mutant HER2 in mammary epithelial cells activated autocrine transforming growth factor (TGF) β1 signaling through a mechanism involving Rac1 and JNK-AP1-dependent transcription. Cells transformed by an activating mutant of H-Ras (G12V) also expressed higher TGF-β1 level through Rac1 activation. In addition, mutant HER2 induced the EGFR ligands TGF-α and amphiregulin at the mRNA and protein levels. Vascular endothelial growth factor (VEGF), a target of the TGF-β-Smad transcriptional regulation, was also induced as a result of expression of mutant HER2. Inhibition of TGF-β signaling with the Alk5 small molecule inhibitor LY2109761 reduced growth and invasiveness of cells expressing mutant HER2. Combined inhibition of intracellular and paracrine effects of mutant HER2 by trastuzumab and the EGFR antibody cetuximab was more efficient than single-agent therapies. These data suggest that mutations in oncogenes such as HER2 and Ras not only alter intracellular signaling and also influence on other components of the tumor microenvironment by inducing several pro-invasive growth factors. In turn, these serve as extracellular targets of novel therapeutic strategies directed at both cancer-driving oncogenes and the modified tumor microenvironment.
doi:10.1038/onc.2010.112
PMCID: PMC2883631  PMID: 20383197
HER2/ErbB2; Ras; TGF-β; VEGF; EGFR ligands; tumor microenvironment
16.  Quality of Life after Autologous Peripheral Blood Stem Cell Transplantation and High-Dose Chemotherapy in High-Risk Breast Cancer Patients 
Breast Care  2009;4(6):379-386.
Summary
Background
As long-term survivors of breast cancer after autologous peripheral blood stem cell transplantation (ASCT) are becoming more numerous, studies addressing the issue of long-term follow-up are necessary. In this study, we report on the quality of life (QOL) after ASCT and high-dose chemotherapy (HDCT).
Patients and Methods
The QOL questionnaire version 3.0 by the European Organization for Research and Treatment of Cancer (EORTC QLQ-C30 version 3.0) was filled in by patients and healthy controls at 5 time points. After obtaining the results, we analyzed the correlation between QOL and the effect factors.
Results
Some functions got significantly worse, and some symptoms got more serious after ASCT and HDCT. However, most of them improved with time and were comparable to the healthy controls after 5 years. QOL was in part related to age, tumor characteristics, educational level, marriage status, and income.
Conclusions
Evaluating QOL allows medical workers to fully understand a patient's state of health, and aid the estimation and selection of clinical treatment methods as well as improve recovery.
doi:10.1159/000266759
PMCID: PMC2942001  PMID: 20877673
Quality of life; Autologous peripheral blood stem cell transplantation; High-risk breast cancer
17.  Inhibition of pleural metastasis of collecting duct carcinoma of the kidney by modified cytokine-induced killer cells: A case report and review of the literature 
Oncology Letters  2010;1(6):955-958.
Collecting duct carcinoma (CDC) or Bellini duct carcinoma of the kidney is a rare, but highly aggressive renal epithelial malignancy, with an extremely poor prognosis. Modified cytokine-induced killer (mCIK) cells were injected into the pleural cavity to treat pleural metastasis of CDC. The patient, a 33-year-old male, was admitted to hospital for further treatment for severe pleural metastasis of CDC. We cured the pleural metastasis through intrapleural infusion with mCIK cells. After receiving this innovative treatment, the patient exhibited a positive response: the cough, dyspnea, chest distress and thoracalgia were evidently relieved, while the pleural fluid became clear after exhibiting haematodes and its level decreased significantly. The patient achieved partial success. This novel immunotherapy method is a promising treatment for patients with refractory pleural metastasis.
doi:10.3892/ol.2010.168
PMCID: PMC3412461  PMID: 22870094
collecting duct carcinoma; pleural metastasis; immunotherapy
18.  Exosomes as a tumor immune escape mechanism: possible therapeutic implications 
Advances in cancer therapy have been substantial in terms of molecular understanding of disease mechanisms, however these advances have not translated into increased survival in the majority of cancer types. One unsolved problem in current cancer therapeutics is the substantial immune suppression seen in patients. Conventionally, investigations in this area have focused on antigen-nonspecific immune suppressive molecules such as cytokines and T cell apoptosis inducing molecules such as Fas ligand. More recently, studies have demonstrated nanovesicle particles termed exosomes are involved not only in stimulation but also inhibition of immunity in physiological conditions. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules such as PD-1L and FasL. Concentrations of exosomes from plasma of cancer patients have been associated with spontaneous T cell apoptosis, which is associated in some situations with shortened survival. In this paper we place the "exosome-immune suppression" concept in perspective of other tumor immune evasion mechanisms. We conclude by discussing a novel therapeutic approach to cancer immune suppression by extracorporeal removal of exosomes using hollow fiber filtration technology
doi:10.1186/1479-5876-6-37
PMCID: PMC2504474  PMID: 18644158
19.  Abrogation of TGFβ signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis 
Cancer cell  2008;13(1):23-35.
Summary
Aberrant TGFβ signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells, are recruited into mammary carcinomas with type II TGFβ receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues, and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGFβ1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFβ mediated metastasis through enhancing tumor cell invasion and metastasis.
Significance
TGFβ is a very important tumor suppressor. Inactivation of TGFβ signaling frequently occurs in human cancers and contributes to tumor metastasis. However, the contribution of host cells in this process is unclear. Here, we show that deletion of Tgfbr2 in mammary carcinoma cells results in increased chemokine signals that enhance Gr-1+CD11b+ myeloid cell infiltration into tumors, which leads to enhanced tumor invasion and metastasis. Gr-1+CD11b+ cell infiltration also results in increased TGFβ production in tumors with Tgfbr2 deletion. Thus tumor-suppressing role of TGFβ can be switched to tumor promoting through the recruitment of Gr-1+CD11b+ cells in the tumor microenvironment. Inhibition of Gr-1+CD11b+ cell production/recruitment could improve host immunosurveillance and inhibit tumor metastasis, having the effect of “killing two birds with one stone”.
doi:10.1016/j.ccr.2007.12.004
PMCID: PMC2245859  PMID: 18167337
TGFβ; tumor progression; metastasis; inflammation; tumor microenvironment; bone marrow; myeloid cells; matrix metalloproteinase; chemokine

Results 1-19 (19)