Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer

EGFr/HER1 and c-erbB-2/HER2 expression are associated with poor prognosis in breast cancer. The type I receptor tyrosine kinase (RTK) family to which they belong has four members (HER1-4). In this study, expression of HER1-4 and oestrogen receptor (ER) expression were determined by immunohistochemistry in 220 breast carcinomas. Elevated expression of HER1 was observed in 16.4%, HER2 in 22.8%, HER3 in 17.5%, and HER4 in 11.9% of these tumours. Patients whose tumours overexpressed HER1, 2 or 3 had reduced survival (p= <0.001), whereas those whose tumours overexpressed HER4 had increased survival (p=0.013); 38.6% of cases overexpressed one or more of HER1, 2 or 3. HER4 was rarely overexpressed with other HERs (1.4% of cases). Cox's multiple regression analysis demonstrated that overexpression of HER1/2/3, HER4, and standard prognostic indicators independently affected survival. HER1-3 expression was related to ER negativity (p<0.0001, chi2). Patients with ER-positive, HER1-3-positive tumours had a significantly poorer survival (p<0.001) than those with ER-positive/HER-negative or HER4-positive tumours. Expression of HER RTKs displays complex interactions between different family members. There is a strong interaction, in terms of survival, between HER expression and ER expression. The development of HER-targeted agents (eg Herceptin, Iressa), and agents targeted at the downstream signalling pathways, therefore provides new possibilities in the treatment of breast cancer.     

Soluble fragment of Her2/neu receptor in the serum of patients with breast cancer with different levels of this protein expression in the tumor

Serum level of soluble HER2/neu in patients with tumors characterized by high expression of this protein (2+/3+ according to immunohistochemical analysis) was significantly higher than in patients with low expression of HER2/neu and in women with benign diseases of the mammary glands. The level of HER2/neu in the serum decreased after removal of the primary tumor in the majority of patients. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 427–430, April, 2007     

Biological role of NHERF1 protein expression in breast cancer

Aims:  To determine the role of Na+/H+ exchanger regulatory factor ( NHERF1 ) in breast cancerogenesis and progression.
Methods and results:  NHERF1 expression was examined in normal tissue, ductal carcinoma in situ (DCIS), invasive carcinoma (IBC), synchronous metastatic lymph node and metachronous distant metastases of a retrospective series of breast cancers. Fifty-one IBC, 42 DCIS and normal tissues were examined immunohistochemically, and the colocalization between NHERF1 and HER2/neu was studied by immunofluorescence. NHERF1 showed a different localization and pattern of expression in the different compartments of the breast. The mean value of cytoplasmic NHERF1 expression in paired samples was significantly higher in DCIS, IBC, distant metastases and metastatic lymph nodes with respect to normal tissues. Moreover, in metastatic lymph nodes NHERF1 was exclusively cytoplasmic. In the membrane NHERF1 was colocalized with overexpressed HER2/neu in DCIS, IBC and distant metastases.
Conclusions:  Breast cancerogenesis is characterized by increased cytoplasmic expression of NHERF1 as the tumour progresses, suggesting a role in this process. The switch from apical membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its subcellular localization.     

Breast cancer micrometastases: Different interactions of carcinoma cells with normal and cancer patients' bone marrow stromata

The apparently dormant breast cancer micrometastases in haemopoietic marrow are correlated with distant metastatic carcinoma dissemination. We studied in vitro interactions of carcinoma cells with adjacent stromata, using connective tissue cell cultures from breast and bone marrow samples of normal donors, comparing them to the pericancerous breast tissue and bone marrows of 12 selected patients with invasive breast carcinomas. Cancer cells were detected by immunocytochemistry and RT-PCR in all the bone marrows and in most blood samples of the studied patients. We monitored the growth and interaction of carcinoma MCF-7 cells with the stromata. The normal breast stroma sustained typical massive cancer growth. The pericancerous breast stroma induced the invasive mesenchymal pattern of growth. Normal bone marrow stroma induced the same conversion and was highly adhesive, retaining the cells in the stroma, but carcinoma patients' bone marrow stromata underwent low adhesive interactions with cancer cells, releasing them potentially into the circulation. The semi-quantitative RT-PCR indicated an enhanced expression of the hepatocyte growth factor and its receptor c-met in breast and bone marrow stromata of cancer patients. The input of cancer cells into the normal bone marrow may induce modifications of the local microenvironment, favourable for growth and release of carcinoma cells into the systemic circulation, which correlate with the poor prognosis of patients with bone marrow micrometastases. This revised version was published online in July 2006 with corrections to the Cover Date.     

New insights on the role of CXCR4 in cancer metastasis

The evolutionarily conserved chemokine superfamily encodes small peptide molecules that bind G-protein-coupled receptors. In humans, this superfamily includes at least 46 ligands and 18 receptors. In general, chemokines and their receptors form a chemotactic network that regulates the migration of cells to specific micro-anatomical destinations within an organism in an organized and non-random manner. Recent studies place chemokine-chemokine receptor pairs at the centre of not only physiological cell migration, but also pathological processes such as metastasis. This mini review considers some recent data on how chemokines regulate tumour cells during metastasis. These observations suggest novel ways for pharmacological intervention.     

Mapping the CXCR4 receptor on breast cancer cells

The CXCR4 receptor triggers cell migration and, in breast cancer, promotes metastasis. To date, the dynamic assembly of CXCR4 on the cell surface as a mediator of receptor binding is not well characterized. The objective of this work is to quantify the density, spatial organization, and magnitude of binding of the CXCR4 receptor on live metastatic breast cancer (MBC) cells. We measured the Young's modulus, the CXCR4 surface density, and CXCR4 unbinding force on MBC cells by atomic force microscopy. We conclude that the CXCR4 density, spatial organization, and matrix stiffness are paramount to achieve strong binding.     

骨形态发生蛋白9体外调节乳腺癌细胞与骨髓间充质干细胞的相互作用

 目的 观察骨形态发生蛋白9（BMP9）在共培养条件下对HS-5成骨分化及MDA-MB-231转移相关因子表达的影响。方法 采用Transwell小室间接共培养MDA-MB-231细胞与HS-5细胞,加入BMP9条件培养基,使用化学发光法、细胞化学染色法及茜素红S染色法分别检测HS-5细胞碱性磷酸酶（ALP）活性、ALP表达及细胞钙盐沉积的变化;应用RT-PCR及Western blot观察HS-5细胞骨钙素（OCN）、MDA-MB-231细胞甲状旁腺激素相关蛋白（PTH-rP）及白细胞介素6(IL-6)mRNA和蛋白表达的变化。结果 HS-5的ALP活性升高,并呈时间依赖性,第9天达最高,随后降低;ALP细胞化学染色结果进一步证实这一点;HS-5细胞钙盐沉积增多、OCN mRNA和蛋白的表达升高（P<0.05）;MDA-MB-231转移相关的PTH-rP及IL-6 mRNA和蛋白的表达下降（P<0.05）。结论 BMP9能够调节乳腺癌细胞与骨髓间充质干细胞的相互作用。     

A murine model of bone marrow micrometastasis in breast cancer

Bone marrow (BM) is one of the most common sites and often the first clinical indication of metastatic progression of breast cancer. Multivariate analyses have shown that the presence of cytokeratin positive tumor cells in the marrow of women with newly diagnosed stage I, II or III breast cancer is an independent predictor of survival. The objective of this study was to develop an orthotopic model of spontaneous BM metastasis to facilitate studies of this process. A murine mammary adenocarcinoma cell line, Clone 66, was transduced with the neomycin resistance gene (Cl66^neo) and injected orthotopically into female Balb/c mice. Polymerase chain reaction (PCR) for the neo gene performed on BM cells harvested from tumor bearing mice demonstrated as few as 10² injected tumor cells produced BM micrometastases at 4 weeks post-injection. Small foci of tumor cells were identified in the mammary fatpad (mfp) without gross evidence of primary tumors. Higher doses of tumor cells produced BM micrometastases, detectable by PCR, at one week post-injection. Constructs containing green fluorescent protein (GFP) and the neomycin resistance gene (neo) were also transduced into Clone 66 cells (Cl66-GFP^neo) and injected into the mfp. GFP transduced tumor cells were identified in multiple tissues in addition to BM by flow cytometric analysis (FACS) but less 13% of the animals developed gross metastases. This model is a clinically relevant tool for the analysis of organ specificity of metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor gene that was shown to be deleted in 10% to 20% of breast cancers by array comparative genome hybridization analysis. We developed fluorescent in situ hybridization to detect the ING4 gene directly in the tissue samples on tumor tissue microarrays. We evaluated the ING4 gene status in 1033 breast cancer tissue samples and observed that ING4 was deleted in 16.5% (170/1033) of all breast cancers. ING4 deletion was significantly associated with Her2 overexpression: of the tumors with ING4 deletion, 23.8% (39/164) were human epidermal growth factor 2 (HER2) positive, as compared with 14.1% (115/814) of the tumors without ING4 deletion (P = .002). In addition, the tumors with ING4 deletion were more likely to belong to the HER2 molecular subtype (estrogen receptor negative/progesterone receptor negative/human epidermal growth factor positive) of breast cancer, compared with the other subtypes (28.4% HER2 versus 15.7% all, P = .002). ING4 deletion did not affect survival outcome of all patients with breast cancer (P = .797) or of the patients with HER2-positive tumors (P = .792). We conclude that ING4 deletion in breast cancer is relatively common, as 1 in 6 breast cancer harbors ING4 deletion. Furthermore, ING4 deletion is more prevalent in HER2-positive tumors, suggesting a functional antagonistic relationship between the ING4 tumor suppressor and the HER2 oncogene. These results sustain the view that ING4 is a tumor suppressor in breast cancer and suggest that ING4 deletion may contribute to the pathogenesis of HER2-positive breast cancer.     

Tbet promotes CXCR6 expression in immature natural killer cells and natural killer cell egress from the bone marrow

Tbet-deficient mice have reduced natural killer (NK) cells in blood and spleen, but increased NK cells in bone marrow and lymph nodes, a phenotype that is thought to be the result of defective migration. Here, we revisit the role of Tbet in NK cell bone marrow egress. We definitively show that the accumulation of NK cells in the bone marrow of Tbet-deficient Tbx21^−/− animals occurs because of a migration defect and identify a module of genes, co-ordinated by Tbet, which affects the localization of NK cells in the bone marrow. Cxcr6 is approximately 125-fold underexpressed in Tbx21^−/−, compared with wild-type, immature NK cells. Immature NK cells accumulate in the bone marrow of CXCR6-deficient mice, and CXCR6-deficient progenitors are less able to reconstitute the peripheral NK cell compartment than their wild-type counterparts. However, the CXCR6 phenotype is largely confined to immature NK cells, whereas the Tbet phenotype is present in both immature and mature NK cells, suggesting that genes identified as being more differentially expressed in mature NK cells, such as S1pr5, Cx3cr1, Sell and Cd69, may be the major drivers of the phenotype.     

Low expression of HER2 protein in breast cancer is biologically significant

HER2 status is routinely tested using immunohistochemistry or FISH following the licensing of a therapeutic agent targeting HER2. However, neither of these methods provides quantitative information relating to the 70-80% of patients with levels of expression lower than the assay detection thresholds. In this study, radioimmunohistochemistry was used to detect quantitative HER2 protein expression in 178 breast cancers. Survival analysis was performed, as were correlations with known prognostic variables and with overexpression of other HER family members. It is demonstrated that the populations expressing very high and very low levels of HER2 are each associated with increased risk of cancer-specific death on survival analysis (p = 0.0043). The group with low levels of HER2 was more likely to be of higher grade, EGFR-positive and ER/HER3/HER4-negative. HER2-positive cases were frequently ER-negative/HER3-positive, whilst cases with normal HER2 expression were often ER-positive/HER4-positive. The aggressive nature of the tumour group with low HER2 expression may be explained by actions of other HER family members, particularly EGFR, but whether these or other factors have a negative regulatory effect on HER2 expression remains to be determined.     

Identification and validation of DOCK4 as a potential biomarker for risk of bone metastasis development in patients with early breast cancer

Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture-mass spectrometry; SILAC-MS) to compare protein expression in a bone-homing variant (BM1) of the human breast cancer cell line MDA-MB-231 with parental non-bone-homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC-MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone-homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4-shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06–4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176–3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4-expressing tumours. High DOCK4 expression was not associated with metastasis to non-skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Tartrate-resistant acid phosphatase as a marker of bone metastases in patients with breast cancer and prostate cancer

Serum activity of tartrate-resistant acid phosphatase 5b (TRAP 5b) in patients with breast cancer and prostate cancer having bone metastases was much higher than in healthy donors and patients without skeletal injuries. TRAP 5b activity in patients with breast cancer and multiple bone metastases surpassed that in patients with single bone metastases. The mean activity of TRAP 5b and range of enzyme activity in women treated with bisphosphonates were significantly lower than in patients not receiving antiresorptive therapy. Diagnostic sensitivity and specificity of TRAP 5b as a marker of skeletal metastases in patients with breast cancer were 82 and 87%, respectively. In patients with prostate cancer these indexes were 71 and 83.4%, respectively. Detection of this marker in tumor patients holds much promise for early diagnostics of bone metastases, estimation of the severity of skeletal metastases, and monitoring of the efficiency of bisphosphonate therapy. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 7, pp. 91–93, July, 2004     

Tartrate-resistant acid phosphatase as a marker of bone metastases in patients with breast cancer and prostate cancer

Serum activity of tartrate-resistant acid phosphatase 5b (TRAP 5b) in patients with breast cancer and prostate cancer having bone metastases was much higher than in healthy donors and patients without skeletal injuries. TRAP 5b activity in patients with breast cancer and multiple bone metastases surpassed that in patients with single bone metastases. The mean activity of TRAP 5b and range of enzyme activity in women treated with bisphosphonates were significantly lower than in patients not receiving antiresorptive therapy. Diagnostic sensitivity and specificity of TRAP 5b as a marker of skeletal metastases in patients with breast cancer were 82 and 87%, respectively. In patients with prostate cancer these indexes were 71 and 83.4%, respectively. Detection of this marker in tumor patients holds much promise for early diagnostics of bone metastases, estimation of the severity of skeletal metastases, and monitoring of the efficiency of bisphosphonate therapy.     

The potential usefulness of interleukin-2 activated bone marrow cells as an active therapeutic tool against cytomegalovirus infection in a bone marrow transplantation setting

Bone marrow transplantation (BMT) has been used in recent years for the treatment of immunodeficiency disease, aplastic anemia, and leukemia. However, there are a number of serious problems and limitations associated with autologous or allogeneic BMT. One of these is an increase in opportunistic infections, of which cytomegalovirus (CMV) infection is one of the most important. Cytomegalovirus has been associated with more frequent deaths than any other single agent, with no reproducibly successful or therapy currently available. Recently usage of interleukin-2 or immunomodulation has been suggested as a powerful modality to combat infectious disease. In this study we showed that bone marrow activated in interleukin-2 for 2 days has the ability to lyse spleen cells infected for 3 days with murine CMV (acute infection model) or salivary gland cells infected for 7 days (chronic infection model), while nonactivated bone marrow or natural killer (NK) cells showed no such lysis. The majority of activated cells involved in lysis were antiasialo GM ₁ ^– , Thy-1^±, indicating a population of cells other than the natural killer-cell population involved.     

The skeleton as a unique environment for breast cancer cells

Bone is a favored location for several cancer metastases especially breast, prostate and myeloma. This review evaluates various properties of the skeleton that contribute to its successful colonization by breast cancer cells. The first consideration is the unique aspects of the vasculature of metaphyseal bone, which may account for the initial lodging of breast cancer cells in specific regions of the skeleton. Metasphyseal bone, found at the ends of long bone, in ribs and in vertebrae, is comprised of trabecular bone interspersed with marrow and a rich vasculature. The chemotactic factors that arise from bone marrow and bone cells are discussed in terms of cancer cell migration out of the vasculature and entry of cancer cells into the marrow cavity. Once the breast cancer cells have migrated into the metaphysis, they interact both directly and indirectly with bone cells and other cells in the marrow. As tumor growth progresses, functional bone cells are lost, most likely through apoptosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear expression of CXCR4 in tumor cells of non-small cell lung cancer is correlated with lymph node metastasis

The stromal-derived factor 1α (CXCL12)/chemokine receptor CXCR4 system plays an important role in the metastatic process of a variety of cancers, with CXCR4 frequently expressed by tumor cells homing to CXCL12-rich compartments. The current study evaluated a possible association of CXCR4 expression with lymph node metastasis in primary non–small cell lung cancer. CXCR4 expression levels were evaluated using immunohistology in 46 non–small cell lung cancer specimens of patients without or with lymph node involvement (N0 = 24, N1/N2/N3 = 22). Evaluation of immunostaining was performed semiquantitatively by visual assessment. Statistical analyses with multiple testing adjustments for confirmatory comparisons were performed to assess relevant parameters associated with lymph node metastases. In all samples of non–small cell lung cancer, tumor cells stained positively for cytoplasmic CXCR4. The intensity of the CXCR4 staining varied considerably between specimens: 2 (4%) tumors demonstrated weak cytoplasmic CXCR4, 22 (48%) intermediate, and 22 (48%) strong staining. Membranous staining was absent; however, nuclear staining of CXCR4 was observed in 5 non–small cell lung cancer samples. Statistical analyses of the association between presence of lymph node metastases and CXCR4 expression levels revealed that cytoplasmic CXCR4 expression was not associated with the presence of lymph node metastases. However, nuclear CXCR4 was significantly correlated with increasing lymph node stage (P = .008), linear-to-linear association. The association between aberrant expression of CXCR4 in the nucleus of non–small cell lung cancer and metastasis to lymph nodes points toward a potential tumor metastasis promoting function of nuclear CXCR4.     

CXCR4-siRNA表达载体的构建及其对体外乳腺癌细胞侵袭能力的影响

目的 构建趋化因子受体（CXCR4）小分子干扰RNA ( siRNA) 表达载体,研究其对乳腺癌细胞侵袭能力的影响。方法 选择CXCR4高表达的乳腺癌MDA-MB-231细胞株,设计合成人CXCR4基因不同靶点的能编码siRNA的2条双链DNA序列,克隆到真核表达载体pGE-1-U6/kna中构建siRNA表达载体,体外脂质体介导转染MDA-MB-231细胞,用Western blot分析CXCR4蛋白表达,transwell 小室检测细胞的侵袭能力。结果 成功构建了CXCR4-siRNA表达载体,瞬时转染乳腺癌细胞后能显著降低CXCR4的蛋白表达,可有效抑制人类乳腺癌MDA-MB-231细胞的侵袭能力。结论 CXCR4- siRNA表达载体通过降低CXCR4基因的蛋白表达能显著抑制人类乳腺癌细胞的侵袭能力,有望为乳腺癌转移的基因治疗开辟新途径。