Higher Expression Of p-ERK Research Articles

Zinc attenuates arsenic overdose-induced brain damage via PERK/ATF6 and TLR/MyD88/NF-κB pathways

Exposure to arsenic (As), a widespread non-metallic toxicant in nature, often results in neurotoxicity, although the exact mechanism is unknown. Zinc (Zn) is a powerful nutrient often thought to be beneficial for growth, development and immunity. Whether Zn can rescue brain damage caused by As contamination remains to be demonstrated. Therefore, in this study, a 30-day model of As poisoning (2.83 mg/L) in carp was established and treated with Zn (1 mg/L) to investigate the detoxification mechanism involved. Histological observations showed that As induced the loosening of the molecular layer structure of the cerebellum and the dissolution or even disappearance of nuclei, accompanied by the occurrence of microthrombi in the granular layer, and the addition of Zn attenuated such As-induced damage. Further mechanistic studies indicated that Zn ameliorated As exposure-induced abnormalities in antioxidant capacity (decreased CAT and Cu/Zn-SOD), activation of the Nrf2/keap1 pathway and endoplasmic reticulum stress (ERs), which is a key factor in As-induced brain damage. ERs (high expression of PERK, ATF6, CHOP, eiF2α and GRP78) and inflammation (overexpression of TLR2, TLR4, MyD88, IKK, NF-κB, IL-1β and IL-6 and low expression of IκBα and IL-10). We suggest that Zn can alleviate excessive As-induced brain damage by attenuating As-induced oxidative stress, PERK/ATF6 and TLR/MyD88/NF-κB pathways. The present study fills in the preventive mechanism of As injury in fish and provides the possibility of prevention and control of As pollution-induced brain tissue injury by Zn rescue.

P-mTOR, p-ERK and PTEN Expression in Tumor Biopsies and Organoids as Predictive Biomarkers for Patients with HPV Negative Head and Neck Cancer

BackgroundSurvival rates of head and neck squamous cell carcinoma (HNSCC) have only marginally improved in the last decades. Hence there is a need for predictive biomarkers for long-time survival that can help to guide treatment decisions and might lead to the development of new therapies. The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is the most frequently altered pathway in HNSCC, genes are often mutated, amplificated and overexpressed causing aberrant signaling affecting cell growth and differentiation. Numerous genetic alterations of upstream and downstream factors have currently been clarified. However, their predictive value has yet to be established. Therefore we assess the predictive value of p-mTOR, p-ERK and PTEN expression.MethodsTissue microarrays (TMA’s) of HPV-negative patients with oropharyngeal (n = 48), hypopharyngeal (n = 16) or laryngeal (n = 13) SCC, treated with primary chemoradiation (cisplatin/carboplatin/cetuximab and radiotherapy), were histologically stained for p-mTOR, PTEN and p-ERK. Expression was correlated to overall survival (OS), disease free survival (DFS) and locoregional control (LRC). Also p-mTOR was histologically stained in a separate cohort of HNSCC organoids (n = 8) and correlated to mTOR-inhibitor everolimus response.ResultsHigh p-mTOR expression correlated significantly with worse OS in multivariate analysis in the whole patient cohort [Hazar Ratio (HR) 1.06, 95%CI 1.01–1.11, p = 0.03] and in the cisplatin/carboplatin group with both worse OS (HR 1.09, 95%CI 1.02–1.16, p = 0.02) and DFS (HR 1.06, 95%CI 1.00–1.12, p = 0,04). p-ERK expression correlated significantly with DFS in univariate analysis in the whole patient cohort (HR 1.03, 95%CI 1.00–1.05, p = 0.04) and cisplatin/carboplatin group (HR 1.03, 95%CI 1.00–1.07, p = 0.04). PTEN-expression did not correlate with OS/DFS/LRC. Better organoid response to everolimus correlated significantly to higher p-mTOR expression (Rs = − 0.731, p = 0.04).ConclusionsHigh p-mTOR expression predicts and high p-ERK expression tends to predict worse treatment outcome in HPV negative HNSCC patients treated with chemoradiation, providing additional evidence that these markers are candidate prognostic biomarkers for survival in this patient population. Also this study shows that the use of HNSCC organoids for biomarker research has potential. The role of PTEN expression as prognostic biomarker remains unclear, as consistent evidence on its prognostic and predictive value is lacking.

Lafoensia pacari A. St.-Hil.: Wound healing activity and mechanism of action of standardized hydroethanolic leaves extract

Ethnopharmacological importanceLafoensia pacari A. St.-Hil., (Lythraceae) is a native tree of Brazilian Cerrado and commonly known in Brazil as "mangava-brava". Its leaves are used in Brazilian folk medicine in wound healing, cutaneous mycoses, and in the treatment of gastritis and ulcers. Aim of the studyThe present study was designed to evaluate the wound healing activity and mechanism of action of the hydroethanolic extract of Lafoensia pacari A. St.-Hil. leaves (HELp), and to advance in its chemical profiling. Materials and methodsHELp was prepared by maceration in 70% hydroethanolic solution (1:10, w/v). The phytochemical analyses were investigated using colorimetry and electrospray ionization/mass spectrometric detection (ESI-MSn). Its in vitro cytotoxicity was evaluated in CHO-K1 and L929 cells, while the in vivo acute toxicity was performed in mice. The potential in vivo wound healing activity was assessed using excision and incision rat models and histopathology of the wounded skin (excision model) was carried out. The in vitro wound healing activity of HELp was demonstrated by scratch assay in L-929 cells, by measuring proliferation/migration rate and p-ERK 1/2 protein expression using western blot analysis. HELp's in vivo anti-inflammatory activity was evaluated by lipopolysaccharide (LPS) induced peritonitis in mice, along with the determination of nitric oxide (NO) and cytokines (TNF-α and IL-10) in the peritoneal lavages. Its potential in vitro antibacterial activity was performed using microbroth dilution assay, while in vitro antioxidant activities was by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and ferric reducing antioxidant power (FRAP) assays. ResultsThe phytochemical analysis of HELp revealed the presence of polyphenols with ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside being the most prominent. HELp showed no toxicity on CHO-k1 and L929 cell lines. Topical treatment with HELp (10 and 30 mg/g of gel) presented increased rates of wound contraction at all the days evaluated with complete wound re-epithelialization at 22.0 ± 1.5 (p < 0.05) and 21.7 ± 1.6 (p < 0.01) days, respectively. Topical application of HELp (10, 30 or 100 mg/g of gel) in incised wounds caused an increase in tensile break strength at all concentrations resulting in moderate re-epithelialization and neovascularization, increased cell proliferation an accelerated remodeling phase of the wound, in a manner comparable to standard drug (Madecassol®, 10 mg/g). In the scratch assay with L929 cells, HELp (0.1 and 0.03 mg/mL) and PDGF (5 ng/mL) resulted in the increased proliferation/migration rate of fibroblasts and higher expression of p-ERK 1/2 protein. In LPS-induced peritonitis, HELp (100 and 200 mg/kg p.o.) decreased total leukocyte migration, comparable to the dexamethasone (0.5 mg/kg p.o.). In RAW 264.7 macrophages activated by LPS, HELp produced anti-inflammatory activity dependent on increased concentrations of IL-10, reduction in NO production, without altering the TNF-α levels. HELp also presented potent antioxidant activity in the DPPH and FRAP, but lacks in vitro antibacterial activity. ConclusionThe present study results support the popular use of the leaves of L. pacari in the treatment of wounds. Its wound healing activity is multi-targeted and involves inhibition of the proliferative and anti-inflammatory phases, antioxidant and positive modulation of the remodeling phase that might be involved different secondary metabolites, with emphasis on the ellagic acid, punicalagin, punicalin, kaempferol, quercetin-3-O-xylopyranoside and quercetin-3-O-rhamnopyranoside.

CD133 mediates the TGF-β1-induced activation of the PI3K/ERK/P70S6K signaling pathway in gastric cancer cells.

Cluster of differentiation (CD)133 has been reported to be involved in the activation of the extracellular signal-regulated kinase (ERK) signaling pathway in different types of cancer cells. CD133 has been reported to be involved in the activation of the ERK signaling pathway in various cancer cells. Transforming growth factor (TGF)-β1 has also been reported to mediate the activation of the ERK signaling pathway. In addition, TGF-β1 has been previously shown to mediate the activation of the ERK signaling pathway. Hence, the present study investigated the function of CD133 in the TGF-β1-induced activation of the ERK/P70S6K signaling pathway in human gastric cancer (GC) cells. To this end, GC cell lines SGC7901 and MKN45 were treated with TGF-β1. The expression of CD133, phospho-ERK (p-ERK) and phospho-P70S6 kinase (p-P70S6K) was upregulated in the cells treated with TGF-β1, while the expression of ERK and P70S6K was not altered. To investigate whether CD133 is involved in the TGF-β1-induced activation of the ERK/P70S6K signaling pathway in GC cells, immunomagnetic cell sorting was employed to isolate CD133+ GC cells, and a CD133-expression construct or CD133-targeting small interfering ribonucleic acids were transfected into cells to modulate the expression of CD133. Subsequently, the expression of CD133, ERK, p-ERK, P70S6K, and p-P70S6K was analyzed by western blotting. The CD133+ cells displayed a high expression of p-ERK and p-P70S6K. Furthermore, SGC7901 GC cells were treated with U0126, an inhibitor of the ERK signaling pathway, to assess whether CD133 is upstream of ERK/P70S6K. The results showed that the expression of p-ERK and p-P70S6K was downregulated in the cells treated with U0126, while the expression of CD133 remained unaltered. The above preliminary results showed that CD133 likely mediates the TGF-β1-induced activation of the ERK/P70S6K signaling pathway in human GC cells. To further understand the mechanism of regulation of the ERK/P70S6K signaling pathway by CD133, the expression of CD133 was modulated by transfecting cells with CD133-expression constructs or CD133-targeting small interfering ribonucleic acids. Results indicated that overexpression and silencing of CD133 directly increased and decreased the expression of p-ERK and p-P70S6K, respectively. Therefore, we hypothesized that CD133 mediates the TGF-β1-induced activation of the PI3K/ERK/P70S6K signaling pathway in human GC cells.

Reversion of anergy signatures in clonal CD21low B cells of mixed cryoglobulinemia after clearance of HCV viremia

AbstractHepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of IgM+CD27+ B cells. These cells display both the features of anergy induced by continual engagement of the B-cell receptor (BCR), such as high expression of phosphorylated extracellular signal–regulated kinase (pERK) and reduced lifespan, and of virus-specific exhaustion, such as CD21low phenotype and a defective response to ligation of BCR and Toll-like receptor 9 (TLR9). MC usually regresses after eradication of HCV with interferon, whose immunomodulatory activity might contribute to this effect. We investigated the phenotypic and functional changes in clonal B cells of MC patients with sustained virologic responses to direct-acting antivirals (DAAs), which lack immunomodulatory properties. We found that high pERK expression and accelerated apoptosis revert within 4 weeks after beginning therapy, whereas clonal B cells unresponsive to TLR9 stimulation persist for at least 24 weeks, although they may partially rescue normal CD21 expression. Thus, similar to mouse models, features of anergy in MC B cells rapidly revert after disengagement from HCV, whereas virus-specific exhaustion imparts a durable inhibitory imprint on cell function. Treatment of HCV+ MC with DAAs provides a valuable tool for untangling the molecular mechanisms of anergy and exhaustion in human B cells.

Targeting inhibition of extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) suppresses growth and angiogenesis of gastric cancer.

AZD6244 (ARRY-142886), a highly selective MAPK-ERK kinase inhibitor, has shown excellent clinical efficacy in many tumors. However, the anti-tumor and anti-angiogenesis efficacy of AZD6244 on gastric cancer has not been well characterized. In this study, high p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. For absence of NRAS, KRAS and BRAF mutation, SGC7901 and BGC823 gastric cancer cells were relative resistance to AZD6244 in vitro. And such resistance was not attributed to the insufficient inhibition of ERK phosphorylation. However, tumor growth was significantly suppressed in SGC7901 xenografts by blockage of angiogenesis. This result was further supported by suppression of tube formation and migration in HUVEC cells after treatment with AZD6244. Moreover, the anti-angiogenesis effect of AZD6244 may predominantly attribute to its modulation on VEGF through p-ERK − c-Fos − HIF-1α integrated signal pathways. In conclusions, High p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. Targeting inhibition of p-ERK by AZD6244 suppress gastric cancer xenografts by blockage of angiogenesis without systemic toxicity. The anti-angiogenesis effect afford by AZD6244 may attribute to its modulation on p-ERK − c-Fos − HIF-1α − VEGF integrated signal pathways.

Targeting RAS-MAPK-ERK and PI3K-AKT-mTOR signal transduction pathways to chemosensitize anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive and chemoresistant tumor with dismal prognosis. Most ATCs harbor mutations that activate RAS/MAPK/ERK and PI3K/AKT/mTOR pathways. Therefore, we investigated and correlated the expression of phosphatase and tensin homolog, pERK, and pAKT proteins as well as mutations of BRAF, RAS, and p53 genes in samples of patients with ATC. Furthermore, we evaluated the potential of inhibition of these pathways on chemosensitization of ATC using 2 thyroid carcinoma cell lines (FRO and SW1736). Our results revealed a negative correlation between the activity of RAS-MAPK-ERK and PI3K-AKT-mTOR pathways in samples of patients. To be specific, the PI3K-AKT-mTOR pathway was suppressed in patients with activated NRAS or high pERK expression. Invitro results suggest that the inhibition of either RAS-MAPK-ERK or PI3K-AKT-mTOR components may confer sensitivity of thyroid cancer cells to classic chemotherapeutics. This may form a basis for the development of novel genetic-based therapeutic approach for this cancer type.

Role of E2F-1 and its involving pathway in esophageal squamous cell carcinoma.

Esophageal cancer is a lethal disease and the optimal therapy remains unclear. Neoadjuvant chemotherapy provides an increased chance of survival; therefore, we attempted to identify potential molecular markers that might improve evaluations of individual responses to therapy. We recruited 109 patients with resectable esophageal squamous cell carcinoma. The patients underwent radical esophagectomy and did not receive any other perioperative treatment. Expression of E2F-1 and molecules involved in its targeted pathways, pERK, Bim, pRb, epidermal growth factor receptor, EZH2 and pAKT, was investigated immunohistochemically. Correlations were observed between E2F-1 and pRb expression; EZH2 expression was significantly correlated with the degree of carcinoma differentiation (P = 0.01). Stage III patients were found to have longer survival if they did not express pERK than if they did (23 months vs. 11 months, P = 0.01). Patients with E2F-1 not expressing pRb were found to have longer survival times than those with E2F-1 who expressed pRb (18.8 months vs. 8.6 months, P = 0.021). Similarly, stage III patients with E2F-1 but not expressing pERK also survived longer than those expressing pERK (23.5 months vs. 3.9 months, P < 0.001). A high expression of pERK was significantly associated with poor survival in patients with locally advanced esophageal cancer. Expression of a combination of molecules, rather than of individual molecules, was more predictive of disease prognosis. E2F-1 and molecules of its targeted pathways may be candidate proteins as markers of chemosensitivity in esophageal cancer patients.

Abstract 2400: Differential phosphorylation of signaling targets as a function of RAS mutation status and tumor histology in non-small cell lung cancer.

Abstract Background: While the importance of RAS and mTOR signaling pathways in non-small cell lung cancer (NSCLC) is well known, the activation status of key components in the pathway as a function of histology and oncogene mutation status has been not completely described. The purpose of this study is to investigate phosphorylation status of signal transduction pathway members and to elucidate their prognostic significance in relation to adenocarcinoma and squamous cell carcinoma. Methods: Phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK), MEK 1 and 2 (pMEK), and mammalian target of rapamycin (pmTOR) were immunohistochemically examined using tissue microarray samples of 269 surgically resected primary NSCLC. Thyroid transcription factor-1 (TTF-1) expression and mutation status of EGFR and KRAS were also evaluated. The relationships between 5-year progression free survival (PFS) and phospho-protein expression were investigated by univariate analyses of log-rank test and multivariate analyses using Cox regression model. Results: A total of 98 adenocarcinomas and 134 squamous cell carcinomas were included in the analyses. In adenocarcinoma, pERK expression was significantly related to pMEK (P = 0.001), pmTOR (P &amp;lt; 0.001), and TTF-1 (P = 0.013) expression. In squamous cell carcinoma, pERK expression was significantly related to pMEK (P &amp;lt; 0.001) expression. EGFR or KRAS mutations were significantly associated with TTF-1 expression (P = 0.014) in adenocarcinoma. However, EGFR and KRAS mutation status were not significantly associated with pERK, pMEK, and pmTOR expressions in adenocarcinoma. By univariate analyses, high pERK expression was associated with better 5-year PFS (P = 0.018) in adenocarcinoma and high pMEK expression was associated with worse 5-year PFS (P = 0.043) in squamous cell carcinoma. In multivariate analyses, pERK (P = 0.040) and pMEK (P = 0.048) expression were independent prognostic factors. Conclusions: There were different patterns of phospho-protein expression in adenocarcinoma and squamous cell carcinoma. pERK expression was a good prognostic factor only in adenocarcinoma and pMEK expression was a poor prognostic factor only in lung squamous cell carcinoma. Citation Format: Sang-Won Um, Jingxiang Chen, Carrie Lee, Norman E. Sharpless, Patrick J. Roberts, D. Neil Hayes. Differential phosphorylation of signaling targets as a function of RAS mutation status and tumor histology in non-small cell lung cancer. [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 2400. doi:10.1158/1538-7445.AM2013-2400

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

BackgroundKRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC.MethodsPre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry.ResultsOf 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status.ConclusionsKRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.

Abstract 4711: The role ERK1/2 and Androgen Receptor phosphorylation at serine 81 in the progression from hormone naive to castrate resistant prostate cancer

Abstract Prostate cancer incidence in the UK is approximately 35,500 cases per year and the second most common cause of male cancer-specific death. The high mortality rate associated with prostate cancer is attributed to the development of castrate resistance prostate cancer (CRPC). Following the development of CRPC there are few therapeutic options with the mean survival period being only 18-24 months. Cell line studies implement ERK1/2 activation in the transition from hormone naïve prostate cancer (HNPC) to CRPC. Recent evidence suggests that ERK kinase activation induces phosphorylation of AR at Serine 81 (AR81) to stimulate prostate cancer cell growth and induce PSA expression (Shigemura K et al Prostate 2009, 69(9) 949-955). This data suggests that the ERK kinase pathway may provide a novel therapeutic target for treatment of CRPC The aim of the current study was to investigate if the relationship between the AR and ERK1/2 is upheld in clinical prostate cancer specimens. A cohort of 55 patients with matched HNPC and CRPC tissue was established with full clinical follow-up. Immunohistochemistry was employed to assess expression of AR, AR81, ERK1/2 and phosphorylated ERK1/2 (pERK). All 55 patients in the cohort were diagnosed with HNPC and subsequently progressed to CRPC. The median age at diagnosis was 70(66-73), median Gleason sum at diagnosis was 8(6-9) and median time to biochemical relapse was 2.3 years. Mean follow-up was 7.5 (4.3-11.7) years, 4 patients were alive at last follow-up, 35 patients died of their disease and 16 patients died of other causes. In the transition of HNPC to CRPC 8% of patients exhibited an increase in AR81 expression and 20% exhibited an increase in ERK1/2 expression. PSA at diagnosis (p=0.034) and high pERK expression in HNPC (p=0.03) were associated with shorter time to biochemical relapse. A negative correlation between AR and ERK1/2 expression was observed in HNPC (R2=-0.383, p=0.004), but no association with AR81 and ERK1/2 or pERK was observed in this stage of disease. Gleason sum (p=0.009), presence of metastasis at diagnosis (p=0.0002), presence of metastasis at relapse (p=0.003) and high ERK1/2 expression in CRPC (p=0.003) were associated with shorter disease specific survival. In CRPC no correlations were observed between AR or AR81 and ERK1/2 or pERK. AR81 expression did however positively correlate with proliferation index as assessed by Ki67 expression (R2=0.339, p=0.025). Combined expression of AR81 and ERK1/2 resulted in a significantly shorter disease specific survival. Whereas, expression of these protein alone (4.2 years vs. 5.4 years) or not at all (7.2 years) resulted in a longer disease specific survival p=0.0006. In conclusion, although no correlations with AR81 and ERK1/2 or pERK were observed in the current study, high expression of AR81 and ERK1/2 combined had a synergistic effect of decreased disease specific survival. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4711.

Isolation and characterization of a highly metastatic hybrid cell line generated between estrogen receptor (ER) negative and ER positive breast cancer cells in mouse bone marrow.

Abstract Abstract #2053 Background: Breast cancer cells in a patient are heterogeneous. How the presence of these heterogeneous cancer cells may contribute to cancer progression is not clear. Experimental evidence indicates that soluble factors produced by aggressive cancer cells may generate metastasis niches for less aggressive cancer cells. In this study, we sought to determine how a growing orthotopic tumor formed by an aggressive ER negative breast cancer cell line might affect the metastatic potential of a less aggressive ER positive breast cancer cell line.&amp;#x2028; Methods and Results: Five-week-old female nude mice were injected orthotopically with metastasis-competent ER negative human breast cancer MDA-MB-231/GFP/Neo cells. After three weeks, metastasis-incompetent human breast cancer ER positive ZR-75-1/GFP/puro cells were inoculated into these mice via intra-cardiac (IC) route and puromycin resistant metastatic ZR-75-1/GFP/puro cells were flushed out of the bone marrow of one mouse and established as a variant cell line called B6. B6 cells have both estrogen-dependent and -independent cells when they were orthotopically injected into female nude mice with or without estrogen supplementation. The cells isolated from an estrogen-dependent tumor were called B6TE and the cells isolated from an estrogen-independent tumor were called B6TC. Further characterization revealed that B6TC cell was more tumorigenic without estrogen supplementation and has a unique metastatic property. More interestingly, B6TC cell was resistant to both puromycin and G418 suggesting it was derived from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. When compared with parental MDA-MB-231/GFP/Neo, the B6TC cells were found to be more metastatic to lung and bone after IC inoculation as detected by GFP imaging, with significant development of paraplegia in the hind limbs after four weeks. More significantly, B6TC mice also developed metastases to brain, which was not observed in the MDA-MB-231/GFP/Neo group. B6TC cell has a low expression of ER alpha and CD24, and high expression of p-ERK, CD44, ALDH. It also expresses vimentin, CXCR4 and Integrin-β1. Its growth is refractory to the anti-estrogens, raloxifene and tamoxifen.&amp;#x2028; Conclusion: Our study indicates that B6 cells generated in the bone marrow were a heterogeneous population of ER positive and ER negative cells with marked differences in their proliferative potential and malignancy. The low ER-expressing B6TC is a novel hybrid cell line generated spontaneously in a metastatic site, which has propensity to metastasize to brain in addition to lung and bone. The expression of CD44 and ALDH indicates that it has stem cell-like features. Further characterization and molecular profiling is underway to reveal the novel biomarkers. This cell line should be a useful model for the investigation of the molecular mechanism of brain metastasis and the therapeutic strategies for the treatment of metastatic breast cancer resistant to anti-estrogens. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2053.