The type III TGF-beta receptor suppresses breast cancer progression

The TGF-beta signaling pathway has a complex role in regulating mammary carcinogenesis. Here we demonstrate that the type III TGF-beta receptor (TbetaRIII, or betaglycan), a ubiquitously expressed TGF-beta coreceptor, regulated breast cancer progression and metastasis. Most human breast cancers lost TbetaRIII expression, with loss of heterozygosity of the TGFBR3 gene locus correlating with decreased TbetaRIII expression. TbetaRIII expression decreased during breast cancer progression, and low TbetaRIII levels predicted decreased recurrence-free survival in breast cancer patients. Restoring TbetaRIII expression in breast cancer cells dramatically inhibited tumor invasiveness in vitro and tumor invasion, angiogenesis, and metastasis in vivo. TbetaRIII appeared to inhibit tumor invasion by undergoing ectodomain shedding and producing soluble TbetaRIII, which binds and sequesters TGF-beta to decrease TGF-beta signaling and reduce breast cancer cell invasion and tumor-induced angiogenesis. Our results indicate that loss of TbetaRIII through allelic imbalance is a frequent genetic event during human breast cancer development that increases metastatic potential.     

The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.     

Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.     

The breast tumor microenvironment: role in cancer development,progression and response to therapy

Introduction: Numerous clinical and pre-clinical studies have provided ample evidence supporting that the tumor microenvironment plays a significant role during breast cancer development, progression and in determining the therapeutic response.

Areas covered: This review focuses on the evolving concept of the microenvironment as the critical participant in each step of the multi-stage process of malignant progression. Currently, only a small number of molecules form part of routine molecular diagnostics in breast caner, but microenvironment-derived biomarkers are potential additions to existing predictive and prognostic marker panels. The authors discuss the dependency of the breast tumor cells on different components of the microenvironment for their survival, dissemination, dormancy and establishment in secondary sites to form overt metastasis, as well as the potential as a therapeutic target to improve breast cancer outcome.

Expert commentary: Despite the importance in the development of breast cancer, the contribution of the microenvironment is not considered in routine diagnostic testing or informing therapeutic decisions. However, introduction of immunotherapy will increasingly require patient selection based on the stromal composition of the primary breast tumor. Better understanding of the role of specific microenvironment-derived molecules is likely to inform personalized therapy, leading to improved patient outcome.     

抑癌基因IL-24在乳腺癌细胞中的表达及意义

目的研究hIL-24（人白细胞介素24）对MDA-MB-231乳腺癌细胞的生长抑制作用。方法将pDC316-hIL-24-EGFP转染人乳腺癌MDA-MB-231细胞,用RT-PCR法、Western blot法检测IL-24基因在肿瘤细胞中的表达。结晶紫染色法检测IL-24基因的表达对乳腺癌细胞的生长抑制。RT-PCR检测侵袭、转移相关基因的转录。ELISA法检测VEGF、Ang-1的分泌水平。结果 IL-24基因可在MDA-MB-231细胞中成功转录及表达,并对乳腺癌细胞增殖有明显抑制作用。能明显下调MMP-2、MMP-9的转录。VEGF、Ang-1表达下降。结论 IL-24对乳腺癌细胞有明显的抑制生长、侵袭转移、血管生成的作用。     

Rapamycin inhibits multiple stages of c-Neu/ErbB2 induced tumor progression in a transgenic mouse model of HER2-positive breast cancer

Amplification of the HER2 (ErbB2, c-Neu) proto-oncogene in breast cancer is associated with poor prognosis and high relapse rates. HER2/ErbB2, in conjunction with ErbB3, signals through the Akt/phosphatidylinositol 3-kinase pathway and leads to the activation of mammalian target of rapamycin (mTOR), a critical mRNA translation regulator that controls cell growth. Gene expression analysis of mammary tumors collected from mouse mammary tumor virus-c-Neu transgenic mice revealed that mRNA levels of several mTOR pathway members were either up-regulated (p85/phosphatidylinositol 3-kinase and p70S6 kinase) or down-regulated (eIF-4E-BP1) in a manner expected to enhance signaling through this pathway. Treatment of these mice with the mTOR inhibitor rapamycin caused growth arrest and regression of primary tumors with no evidence of weight loss or generalized toxicity. The treatment effects were due to decreased proliferation, associated with reduced cyclin D1 expression, and increased cell death in primary tumors. Whereas many of the dead epithelial cells had the histopathologic characteristics of ischemic necrosis, rapamycin treatment was not associated with changes in microvascular density or apoptosis. Rapamycin also inhibited cellular proliferation in lung metastases. In summary, data from this preclinical model of ErbB2/Neu-induced breast cancer show that inhibition of the mTOR pathway with rapamycin blocks multiple stages of ErbB2/Neu-induced tumorigenic progression.     

Classifying circulating tumor cells to monitor cancer progression

Introduction: The term ‘liquid biopsy’ refers to molecular analysis of a tumor’s genetic features based on circulating genetic material in the peripheral blood derived from circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and circulating miRNAs, and has emerged as a minimally invasive tool in early cancer diagnosis and disease monitoring. CTCs are believed to originate from the primary tumor and obtain genetic heterogeneity during evolution.

Areas covered: The presence of CTCs has been associated with poor clinical outcome in patients with metastatic breast cancer, lung cancer, colorectal cancer and prostate cancer. In addition, the detection of CTCs in patients with early breast cancer has been shown to represent an independent prognostic factor associated with an unfavorable clinical outcome. Moreover, the longitudinal evaluation of CTCs in patients with early breast cancer may reveal the presence of chemo- and hormone-therapy resistant CTCs which are associated with an increased risk for disease relapse and disease-related death.

Expert commentary: The molecular characterization of CTCs may provide an important tool for the monitoring and the evaluation of treatment efficacy in patients with different tumor types such as breast, prostate, colon, and non-small cell lung cancer.     

Stromal oncostatin M cytokine promotes breast cancer progression by reprogramming the tumor microenvironment

The tumor microenvironment (TME) is reprogrammed by cancer cells and participates in all stages of tumor progression. The contribution of stromal cells to the reprogramming of the TME is not well understood. Here, we provide evidence of the role of the cytokine oncostatin M (OSM) as central node for multicellular interactions between immune and nonimmune stromal cells and the epithelial cancer cell compartment. OSM receptor (OSMR) deletion in a multistage breast cancer model halted tumor progression. We ascribed causality to the stromal function of the OSM axis by demonstrating reduced tumor burden of syngeneic tumors implanted in mice lacking OSMR. Single-cell and bioinformatic analysis of murine and human breast tumors revealed that OSM expression was restricted to myeloid cells, whereas OSMR was detected predominantly in fibroblasts and, to a lower extent, cancer cells. Myeloid-derived OSM reprogrammed fibroblasts to a more contractile and tumorigenic phenotype and elicited the secretion of VEGF and proinflammatory chemokines CXCL1 and CXCL16, leading to increased myeloid cell recruitment. Collectively, our data support the notion that the stromal OSM/OSMR axis reprograms the immune and nonimmune microenvironment and plays a key role in breast cancer progression.     

乳腺癌转移抑制基因、尿激酶型纤溶酶原激活剂在乳腺癌组织中的表达

目的探讨乳腺癌组织中乳腺癌转移抑制基因(BRMS1)蛋白和尿激酶型纤溶酶原激活剂(u PA)表达及其临床意义。方法采用免疫组化(Envision二步法)检测90例乳腺癌组织和30例癌旁组织中BRMS1、u PA的蛋白表达情况,并结合临床病理特征分析其临床意义。结果乳腺癌组织中BRMS1和u PA的阳性表达率分别为45.56%和60.00%,乳腺癌旁正常乳腺组织中BRMS1和u PA的阳性表达率分别为93.33%和20.00%,两组比较,差异均有统计学意义(χ2分别=21.02、14.40,P均<0.05);常规苏木精-伊红染色和免疫组化染色显示,BRMS1蛋白表达于细胞核和细胞质内,u PA蛋白阳性表达主要定位于肿瘤细胞胞质。阳性细胞染色呈棕黄色细颗粒状。BRMS1蛋白和u PA表达在TNM分期中Ⅰ期和Ⅱ期乳腺癌中的表达阳性率明显高于Ⅲ期者,差异有统计学意义(χ2分别=5.64、6.13、9.46、36.75,P<0.05);BRMS1和u PA表达在乳腺癌淋巴结转移之间比较,差异均有统计学意义(χ2分别=9.64、36.75,P均<0.05);Spearman相关性分析显示,在乳腺癌组织中,BRMS1蛋白表达与u PA呈明显负相关(r=-0.75,P<0.05)。结论 BRMS1与u PA的异常表达可能参与了乳腺癌的浸润转移,两者在转移的信号转导中可能有一定的相关性。     

抑癌基因PTEN介导的信号转导对乳腺癌生物性状的影响

目的探讨第10号染色体上缺失的与张力蛋白同源的基因（PTEN）对乳腺癌细胞生物性状的影响及其潜在机制。方法利用脂质体介导法将pBP—wt—PTEN、pBP—G129R—PTEN和pBP—G129E—PTEN质粒分别转染入PTEN基因缺失的乳腺癌细胞株ZR-75-1。通过侵袭实验比较pBP—wt—PTEN、pBP—G129R—PTEN和pBP—G129E质粒成功转染细胞和未经转染细胞之间的侵袭能力差异。免疫印迹试验（Western—blot）检测各组细胞的总酪氨酸激酶（FAK）和P^397-FAK磷酸化水平;逆转录-聚合酶链反应（RT—PCR）检测各组细胞p53mRNA水平,流式细胞术分析其凋亡率。结果pBP—wt—PTEN、pBP—G129R-PTEN和pBP—G129E—PTEN 3种质粒转染侵袭抑制率分别为70．4％、6．9％和63．5％;pBP—wt—PTEN细胞P^397-FAK水平比pBP—G129R—PTEN细胞低而与pBP—G129E—PTEN细胞差异无统计学意义（P〉0．05）。pBP—wt—PTEN细胞p53 mRNA水平和凋亡率高于pBP—G129E—PTEN细胞,pBP—G129E—PTEN细胞高于pBP—G129R—PTEN细胞,但pBP—G129R—PTEN细胞与未经转染细胞间差异无统计学意义（P〉0．05）。结论PTEN基因对乳腺癌转移和凋亡有一定的影响。     

CD133-targeted paclitaxel delivery inhibits local tumor recurrence in a mouse model of breast cancer

Expression of the membrane protein CD133 marks a subset of cancer cells with drug resistant phenotype and enhanced tumor initiating ability in xenotransplantation assays. Because drug resistance and tumor relapse are significant problems, approaches to eliminate these cells are urgently needed. As a step towards achieving this goal, we developed polymeric nanoparticles targeting CD133 by conjugating an anti-CD133 monoclonal antibody to nanoparticles formulated using poly(D,L lactide-co-glycolide) polymer. Nanoparticles were loaded with paclitaxel, a microtubule-stabilizing anticancer agent, as well as with 6-coumarin, a fluorescent probe. CD133-targeted nanoparticles (CD133NPs) were efficiently internalized by Caco-2 cells, which abundantly express CD133 (> 9-fold higher uptake than non-targeted control nanoparticles). The effectiveness of CD133NPs in reducing tumor initiating cell (TIC) fraction was investigated using mammosphere formation and soft-agar colony formation assays. Free paclitaxel treatment was not effective in decreasing the TIC population relative to untreated control, whereas CD133NPs effectively decreased the number of mammospheres and colonies formed. In vivo studies in the MDA-MB-231 xenograft model showed that free paclitaxel was initially effective in inhibiting tumor growth but the tumors rebounded rapidly once the treatment was stopped. Tumor regrowth was significantly lower when paclitaxel was delivered through CD133NPs (tumor volume was 518.6 ± 228 vs. 1370.9 ± 295 mm³ for free paclitaxel at 63 days; P < 0.05). Our studies thus show that encapsulation of paclitaxel in CD133NPs results in a significant decrease in the TIC population and improved therapeutic efficacy compared to that with free paclitaxel treatment. These results indicate the potential of targeting anticancer therapeutics to CD133 + cells for reducing tumor recurrence.     

Methotrexate exacerbates tumor progression in a murine model of chronic myeloid leukemia

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). 32Dp210 tumor cells were found to be sensitive to methotrexate (MTX) in vitro, whereas cells expressing the tyrosine-22 DHFR gene were protected from MTX at up to micromolar concentrations. MTX administered at low dose (2 mg/kg/day) did not protect normal C3H-He/J mice from 32Dp210 tumor infused intravenously, with drug toxicity limiting the administration of higher doses. Animals engrafted with transgenic tyrosine-22 DHFR marrow were protected from greater MTX doses (up to 6 mg/kg/day). However, the increased doses of MTX afforded by drug-resistance gene expression surprisingly resulted in decreased survival of the transplanted tumor-bearing animals, with increased levels of tumor detected in peripheral blood. This apparent exacerbation of tumor progression by MTX was not observed in DHFR transgenic mice in which all cells and tissues contain the drug-resistance gene. This suggests that increased tumor progression in MTX-administered animals resulted from MTX sensitivity of a nonhematopoietic host component, thus allowing tumor expansion. We conclude that MTX exacerbates tumor progression in the 32Dp210 model of CML, and that based on this model alternate DHFR inhibitors combined with drug-resistant DHFR or other chemotherapeutic agent/drug-resistance gene combinations may be required for the application of drug-resistance gene expression to the treatment of CML.     

Ovarian cancer cell-derived migration inhibitory factor enhances tumor growth, progression, and angiogenesis

In view of our previous findings that tumor cell-derived macrophage migration inhibitory factor (MIF) increased macrophage-mediated ovarian cancer cell invasiveness in vitro, we investigated the wider significance of ovarian cancer cell-derived MIF for tumor growth, metastasis, and angiogenesis. We found that MIF is expressed in borderline and malignant ovarian tumors, and active MIF is found in malignant ascitic fluid. We next investigated the expression and function of MIF in a syngeneic ovarian cancer model. Stable knockdown of MIF in the murine ovarian cancer cell line ID8 decreased in vivo tumor burden and overall survival. Tumors arising from MIF knockdown cells had decreased proliferation and significantly increased apoptosis. This was associated with an increased phosphorylation of p53 and reduced Akt phosphorylation. MIF knockdown led to a changed cytokine profile in the ascitic microenvironment; tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-10 expression were all significantly decreased. Accompanying this decrease in cytokine expression was a significant decrease in macrophage infiltration into ascites. Additionally, MIF knockdown reduced the expression of proangiogenic cytokines vascular endothelial growth factor and keratinocyte chemoattractant (KC) and reduced the amount of endothelial cells in the malignant ascites. We conclude that autocrine production of MIF by ovarian cancer cells stimulates other cytokines, chemokines, and angiogenic factors that may promote colonization of the peritoneum and neovascularization of tumor deposits.     

Steroid receptors in human breast tumorigenesis and breast cancer progression.

Steroid hormones, in particular estrogen and progesterone, play important roles in normal and neoplastic breast development. Alterations in both estrogen signaling and progesterone signaling likely occur during breast tumorigenesis and breast cancer progression. This is demonstrated by alteration of estrogen (ER) and progesterone (PR) receptor isoform expression as well as other factors such as coregulators, that can affect the activity, directly or indirectly, of in particular ER signal transduction pathways during breast tumorigenesis and breast cancer progression. A commonly emerging theme is the marked alteration of estrogen action that occurs during these processes. Since targeting ER signaling previously was successful, a better knowledge of all the molecular players involved in regulating estrogen signaling pathways and identifying changes that occur in vivo, seems critical to further exploit this previously successful approach and identify new targets for prevention and treatment of human breast cancer.