Hepatic nonparenchymal cells drive metastatic breast cancer outgrowth and partial epithelial to mesenchymal transition

Nearly half of breast carcinoma metastases will become clinically evident five or more years after primary tumor ablation. This implies that metastatic cancer cells survived over an extended timeframe without emerging as detectable nodules. The liver is a common metastatic destination, whose parenchymal hepatocytes have been shown to impart a less invasive, dormant phenotype on metastatic cancer cells. We investigated whether hepatic nonparenchymal cells (NPCs) contributed to metastatic breast cancer cell outgrowth and a mesenchymal phenotypic shift indicative of emergence. Co-culture experiments of primary human hepatocytes, NPCs or endothelial cell lines (TMNK-1 or HMEC-1) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Exposure of carcinoma cells to NPC-conditioned medium isolated soluble factors contributing to outgrowth. To elucidate outgrowth mechanism, epidermal growth factor receptor (EGFR) inhibition co-culture experiments were performed. Flow cytometry analyses and immunofluorescence staining were conducted to quantify breast cancer cell outgrowth and phenotype, respectively. Outgrowth of the MDA-MB-231 cells within primary NPC co-cultures was substantially greater than in hepatocyte-only or hepatocyte+NPC co-cultures. MCF-7 cells co-cultured with human NPCs as well as with the endothelial NPC subtypes grew out significantly more than controls. MCF-7 cells underwent a mesenchymal shift as indicated by spindle morphology, membrane clearance of E-cadherin, and p38 nuclear translocation when in HMEC-1 co-culture. HMEC-1-conditioned medium induced similar results suggesting that secretory factors are responsible for this transition while blocking EGFR blunted the MCF-7 outgrowth. We conclude that NPCs in the metastatic hepatic niche secrete factors that can induce a partial mesenchymal shift in epithelial breast cancer cells thus initiating outgrowth, and that this is in part mediated by EGFR activation. These data suggest that changes in the parenchymal cell and NPC ratios (or activation status) in the liver metastatic microenvironment may contribute to emergence from metastatic dormancy.     

Circulating tumor cells in metastatic breast cancer

BACKGROUND.

The aim of the current study was to assess the prognostic value of baseline circulating tumor cells (CTCs) in a large cohort of patients with newly diagnosed metastatic breast cancer (MBC).

METHODS.

This retrospective study included 185 patients with newly diagnosed MBC evaluated between 2001 and 2007. CTCs were isolated and enumerated before patients started first‐line treatment using the CellSearch system. Overall survival (OS) was calculated from the date of CTC measurement, estimated by the Kaplan‐Meier product limit method, and compared between groups with the log‐rank test. Cox proportional hazards models were fitted to determine the association between CTC levels and OS after controlling for other prognostic factors.

RESULTS.

The median age of the patients at the time of MBC diagnosis was 49 years. Fifty‐six (30.3%) patients presented with de novo metastatic disease, and 129 (69.7%) presented with newly recurrent breast cancer. A total of 114 patients (61.6%) had CTC <5, and 71 (38.4%) had CTC ≥5. The median OS was 28.3 months and 15 months (P < .0001) for patients with CTC <5 and CTC ≥5, respectively. Superior survival among patients with CTC <5 was observed regardless of hormone receptor and HER‐2/neu status, site of first metastases, or whether the patient had recurrent or de novo metastatic disease. In the multivariate model, patients with CTC ≥5 had a hazards ratio of death of 3.64 (95% confidence interval, 2.11‐6.30) compared with patients with CTC <5.

CONCLUSIONS.

The results of this large retrospective study confirms that CTCs are a strong independent predictor of survival among women with either de novo or newly recurrent MBC. CTCs should be considered as a new stratification method for women with newly diagnosed MBC. Cancer 2008. © 2008 American Cancer Society.     

Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells

Introduction  

The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44).     

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Introduction  

Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.     

Circulating tumor cells in metastatic inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer. The aim of this study was to assess the prognostic value of baseline CTCs in metastatic IBC patients.Patients and methodsThis retrospective study included 42 metastatic IBC and 107 metastatic non-IBC patients treated with first- or second-line chemotherapy from January 2004 to December 2007 at MD Anderson Cancer Center. CTCs were detected and enumerated before patients started chemotherapy using the CellSearch™ system.ResultsTen (23.8%) IBC patients versus 48 (44.9%) non-IBC patients had baseline CTCs ≥5 per 7.5 ml of peripheral blood. IBC patients had a lower mean ± SEM CTCs than non-IBC patients (7.6 ± 2.9 versus 34.2 ± 9.1; P = 0.02). The estimated median overall survival was 26.5 versus 18.3 months (P = 0.68) in IBC patients and 37.4 versus 18.3 months (P = 0.016) in non-IBC patients with CTCs <5 and CTCs ≥5, respectively.ConclusionsMetastatic IBC patients had a lower prevalence and fewer CTCs in comparison to metastatic non-IBC patients. Survival of metastatic IBC patients with <5 CTCs was not significantly better than that of patients with ≥5 CTCs. Further research is warranted with prospective assessment of CTCs in IBC patients and their biological characterization.     

转移性乳腺癌循环肿瘤细胞的上皮间质转化水平对化疗疗效的预测意义

目的 检测转移性乳腺癌患者外周血循环肿瘤细胞（CTCs）不同亚型相关基因的表达,探讨其对化疗疗效的预测意义。方法 应用免疫磁性分选（MACS）技术联合逆转录 聚合酶链反应（RT-PCR）法检测58例转移性乳腺癌患者和10例健康人外周血中CTCs上皮型标志物上皮角蛋白（CK18、CK19）和间质型标志物（波形蛋白、纤连蛋白）mRNA的表达。分析上皮型标志物及间质型标志物表达与不同亚型乳腺癌之间的关系,并分别评估具有不同表型CTCs患者之间疗效的差异。结果 在10例健康志愿者的血液样本中,未检测出CK18、CK19、波形蛋白和纤连蛋白mRNA的表达。在58例转移性乳腺癌患者的血液样本中,检测出36例（62.1％）上皮角蛋白表达,19例（32.8％）间质型标志物表达。Luminal A组和HER-2阳性组的上皮型标志物阳性表达率高于三阴性乳腺癌组（P=0.008）,而间质型标志物阳性表达率则低于三阴性乳腺癌组（P＜0.001）。根据不同标志物的表达情况,将患者分为CKs+／EMT-组、CKs-／EMT-组、CKs+／EMT+组和CKs-／EMT+组,4组有效率依次为76.7％、55.6％、33.3％和15.4％,差异有统计学意义（P=0.002）。间质型标志物阴性者的化疗有效率高于间质型标志物阳性者（71.8% vs. 15.8%,P=0.000）。结论 转移性乳腺癌患者中部分CTCs将发生上皮间质转化而丢失上皮型细胞的表型,获得间质型细胞表型。间质型CTCs因具有更强的抵抗化疗药物的能力而存活,这可能是三阴性乳腺癌疗效不佳的原因之一。     

Inhibition of apoptosis in human breast cancer cells: role in tumor progression to the metastatic state

Reduction in apoptosis has been associated with tumor metastases and response to chemotherapy in breast cancer. We examine the influence of apoptosis status and the expression of antiapoptotic proteins Bcl-2 and Bcl-x(L) on metastatic progression and response to therapy in an experimental model of breast cancer. We used human breast cancer cells (MDA-MB 435, MDA-MB 468 and MCF-7) to induce orthotopic xenograft tumors in nude mice. The overexpression of Bcl-2 or Bcl-x(L) influenced tumorigenicity, 468 transfectants being less tumorigenic than control (p < 0.0001). Lung metastasis appeared at day 120 in animals injected with 435/Bcl-2 or 435/Bcl-x(L) and they showed higher metastatic activity than control 435/Neo tumors (p = 0.02). In contrast, mice with 468 tumors were followed for 1 year after tumor excision, but they did not develop metastatic foci. 435/Bcl-2 and 435/Bcl-x(L) transfectant cells bound less readily to laminin (ANOVA, p < 0.0001), fibronectin (ANOVA, p < 0.0001) and collagen type-IV (ANOVA, p < 0.0001) than 435/Neo cells. The overexpression of antiapoptotic proteins in 435 transfectants rescued 20-40% of cells from anoikis at 64 hr in rocking conditions. In contrast, at this time only 5-10% of 468 and MCF-7 transfectant cells were rescued. Thus, the overexpression of the Bcl-2 or Bcl-x(L) associated with the loss of apoptosis in breast cancer cells in vivo may account for their metastatic behavior. These genes increase tumor metastasis when the oncogenic background has triggered the metastatic process, in which anoikis might determine tumor progression when the life span of the cells is extended.     

Phosphatidylcholine-specific phospholipase C activation in epithelial ovarian cancer cells

Elucidation of the mechanisms responsible for aberrant phosphatidylcholine (PC) metabolism in cancer cells may allow identification of novel biomarkers of tumor progression and design of new targeted anticancer therapies. We recently reported up-regulation of PC-specific phospholipases in epithelial ovarian cancer cells (EOC) compared with nontumoral (normal or immortalized) counterparts (EONT). In the present study, we focused, in the same cell systems, on levels, subcellular localization, and activity of PC-specific phospholipase C (PC-PLC), for which a key role in cell proliferation, differentiation, and apoptosis has been shown in several mammalian cells. A 66-kDa PC-PLC isoform, detected in nuclear and cytoplasmic compartments of both EOC and EONT cells, accumulated on the external plasma membrane of cancer cells only, where it colocalized with beta1 integrin, in nonraft membrane domains. PC-PLC activity was 3-fold higher in total cell lysates and 5-fold higher in membrane-enriched fractions of EOC compared with EONT cells. Serum deprivation induced in EOC, but not in EONT, cells a 3-fold decrease in PC-PLC activity, associated with a 40% drop in S-phase fraction. The recovery of both variables to their original levels in serum-restimulated (or lysophosphatidic acid-restimulated) EOC cells was strongly delayed, for at least 24 h, in the presence of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609). The S-phase of serum-restimulated EONT cells was not sensitive to D609. These findings warrant further investigations on the role of PC-PLC and on the effects of its inhibition on the pathways responsible for constitutive EOC cell stimulation and cell proliferation.     

Phloroglucinol suppresses metastatic ability of breast cancer cells by inhibition of epithelial‐mesenchymal cell transition

Metastasis is a challenging clinical problem and the primary cause of death in breast cancer patients. However, there is no therapeutic agent against metastasis of breast cancer cells. Here we report that phloroglucinol, a natural phlorotannin component of brown algae suppresses metastatic ability of breast cancer cells. Treatment with phloroglucinol effectively inhibited mesenchymal phenotypes of basal type breast cancer cells through downregulation of SLUG without causing a cytotoxic effect. Importantly, phloroglucinol decreased SLUG through inhibition of PI3K/AKT and RAS/RAF‐1/ERK signaling. In agreement with in vitro data, phloroglucinol was also effective against in vivo metastasis of breast cancer cells, drastically suppressing their metastatic ability to lungs, and extending the survival time of mice. Collectively, our findings demonstrate a novel anticancer activity of phloroglucinol against metastasis of breast cancer cells, implicating its clinical relevance.     

探讨循环肿瘤细胞的上皮间质转化基因Twist对晚期乳腺癌化疗疗效的影响

目的检测转移性乳腺癌患者外周血循环肿瘤细胞上皮间质转化基因Twist的表达水平及化疗疗效, 探讨Twist对晚期乳腺癌化疗效果的预测意义。方法 应用免疫磁性分选技术(MACS)联合逆转录-聚合酶链反应(RT-PCR)方法检测实验组(58例转移性乳腺癌患者)和对照组(10例健康志愿者)外周血中循环肿瘤细胞(CTCs)Twist基因的表达。分析其与乳腺癌临床病理学参数的关系, 并评估CTCs具有不同Twist基因表达的患者之间治疗效果的差异。结果 在对照组的血液样本中, 未检测出Twist mRNA的表达。在实验组的血液样本中, 19例(32.8%)Twist mRNA表达阳性, 39例(67.2%)Twist mRNA表达阴性。在不同的乳腺癌分子亚型中, 三阴性乳腺癌组的Twist mRNA表达明显高于Luminal A组和Her-2阳性组(P＜0.001)。根据患者化疗后疗效评估结果分析, Twist mRNA表达阳性组患者对化疗的有效率低于阴性组(P＜0.05)。结论 部分循环肿瘤细胞表达Twist基因。发生上皮间质转化的循环肿瘤细胞有更强的抵抗化疗药物的能力, 是三阴性乳腺癌疗效不佳的原因之一。     

Prognostic factors affecting treatment results with combination chemotherapy in metastatic breast cancer

Seventy five patients suffering from metastatic breast cancer previously unexposed to chemotherapy were treated with Cyclophosphamide, Methotrexate and 5-fluorouracil (CMF) upon diagnosis of their first relapse. Dose Level I (85% or more of the planned dose) was given to 14 patients (19%); Dose Level II (66% to 84% of the planned dose) to 29 patients (39%); and Dose Level III (65% or less of the planned dose) to 32 patients (42%). Before initiation of treatment, 41 patients (55%) had a good performance status (PS greater than or equal to 80%) and 34 patients (45%) had a poor performance status (PS less than 80%) according to the Karnofsky scale. The overall response rate was 44%, including 17% complete responses. There was no significant correlation between response rates and the dose levels of chemotherapy. However, patients with a good performance status had a higher response rate (61%) compared with patients with a poor performance status (25%; p = 0.0025). The actuarial 3 year survival according to dose levels of CMF was 15%, 41% and 36% for Dose Levels I, II, and III, respectively (p = 0.34), but was 52% for patients with PS greater than or equal to 80% versus 14% for patients with PS less than 80% (p = 0.0001). These data indicate that the general condition of the patient at the time of initiation of CMF chemotherapy, as reflected by the performance status, may be of greater significance for the prediction of the ultimate treatment outcome rather than the total amount of chemotherapy delivered per se.     

Hepatic resection in metastatic breast cancer: results and prognostic factors.

AIMS: Breast cancer liver metastases (BCLM) usually indicate the presence of disseminated cancer with a very poor prognosis. However, systemic treatments now allow control of tumour progression in certain cases. We evaluated, in a group of highly selected patients with stabilization or complete response to systemic therapy, a particular management protocol for medically controlled BCLM: liver surgery. METHODS: Fifty-two patients underwent surgery between May 1988 and September 1997. Results of this strategy are reported, together with analysis of prognostic factors for survival and recurrence in the remaining liver (RRL). RESULTS: The mean number of cycles of chemotherapy, before surgery, was seven (3-24). Resection was considered to be curative in 86% of cases. The median follow-up was 23 months (1-72 months). The survival after surgery, was 86% at 12 months, 79% at 24 months and 49% at 36 months. The 36-month survival rate differed according to the time to onset of BCLM: 45% before versus 82% after 48 months (P=0.023). The RRL rate at 36 months differed according to the lymph node status of the initial breast cancer: 41% for N0-N1 versus 83% for N1b-N2 (P=0.021). CONCLUSIONS: Adjuvant liver surgery allowed discontinuation of chemotherapy in 46% of cases and, in this highly selected patient group, allowed good quality prolonged survival. It could be included in multicentre treatment protocols for controlled BCLM, one arm with prolonged chemotherapy, one with adjuvant liver surgery.