Human Mammary Carcinoma Research Articles

Intraepidermal Glandular Carcinoma of the Nipple (Mammary Paget-like Disease) in 2 Dogs

Mammary Paget disease, characterized by diffuse infiltration of the nipple and areolar epidermis by carcinoma cells, develops in 1% to 3% of human mammary carcinomas. The purpose of this article is to present 2 cases of intraepidermal adenocarcinoma that resembled human mammary Paget disease, histologically and immunohistochemically, in dogs with underlying mammary carcinoma.

Targeting FcαRI on Polymorphonuclear Cells Induces Tumor Cell Killing through Autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.

Antioxidant and antibacterial activities of bark extracts from Commiphora berryi and Commiphora caudata

This study investigates the in vitro antioxidant and antimicrobial activities of eight extracts obtained from the dried barks of Commiphora berryi and Commiphora caudata (Burseraceae). The radical scavenging activity was assessed by 1,1-diphenyl-2-picryl hydrazyl (DPPH) and nitric oxide assays. The methanolic extracts of C. berryi and C. caudata showed significant DPPH radical scavenging activity, with IC50 values of 26.92 and 21.16 µg mL−1, respectively, and low radical scavenging activity against the nitric oxide assay. The antimicrobial activity of the plants was tested against the Gram-positive and Gram-negative bacteria. The ethyl acetate, chloroform and petroleum ether extracts of C. berryi showed good antibacterial activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration (MIC) of 0.26 mg mL−1, whereas the ethyl acetate and methanol extracts of C. caudata showed moderate antimicrobial activity with an MIC of more than 2.0 mg mL−1 against P. aeruginosa compared to the petroleum ether and chloroform extracts, which showed an MIC of 1.1 mg mL−1. The methanolic extracts of C. berryi and C. caudata also showed moderate cytotoxic activity against a human mammary carcinoma cell line (MCF-7), with values IC50 of 82.6 and 88.4 µg mL−1, respectively.

Autocrine Proliferative Effects of hGH Are Maintained in Primary Cultures of Human Mammary Carcinoma Cells

Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines. We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC. Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined. One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not. Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.

Effect of flavonone hesperidin on the apoptosis of human mammary carcinoma cell line MCF-7

The aim of the present study was to investigate the impact of a natural citrus flavonoglycoside hesperidin on the human mammary carcinoma cell line (MCF-7) and its possible role in the induction of apoptosis. Human breast cancer cells were treated with different concentrations of hesperidin and its cytotoxicity effect was measured by MTT method and lactate dehydrogenase leakage assays. Apoptosis was analyzed by light microscopic studies and DNA fragmentation. The mechanism of apoptosis was studied with the expression of p53 and caspase-3 proteins. Glutathione content was also measured in MCF-7 cells after hesperidin treatment. The results revealed that hesperidin significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. Apoptotic morphology was observed in MCF-7 cells treated with hesperidin at the concentration of 60 and 80 μM. DNA fragmentation, accumulation of p53 and expression of caspase-3 proteins were observed in MCF-7 cells after the treatment with hesperidin. The depletion of GSH and an elevation of LDH were observed in MCF-7 cells treated with hesperidin. Therefore in conclusion, hesperidin induced cytotoxicity in MCF-7 cells in vitro. Apoptosis of MCF-7 cells may be due to the DNA damage and expression of apoptotic proteins. Depletion of GSH may be involved in the induction of apoptosis of Michigan cancer foundation-7 cells.

Extracts of red yolk from duck eggs inhibit metastasis of human mammary carcinoma (4T1) cells in mice

The aim of the present study was to investigate the in vivo anti-metastatic activity of the red pigments of red yolk eggs laid by the ducks dieting on Potamogeton cripus L on the mammary carcinoma (4T1). The pigments were extracted with petroleum ether and acetone (2:1, v/v). BALB/c mice were divided into three groups (n=6), fed with the extracts at 150 mg/kg body weight (BW)/day (DEYE-H) or at 50 mg/kg BW/day (DEYE-L) and identical buffer without the extract (control group). The extracts were administered for 34 days. The treatment significantly inhibited the growth of orthotopical 4T1 tumour (DEYE-H vs control, 1:2; DEYE-L vs control, 2:3) and reduced the metastasis of tumour in the lungs (DEYE-H vs control, 4:7; DEYE-L vs control, 5:7), without statistical difference of body weight among the three groups.

Methylation silencing of angiopoietin‐like 4 in rat and human mammary carcinomas

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2'-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced.

Abstract 203: PKR is a doxorubicin sensitivity biomarker for breast cancer

Abstract Breast cancer is the second most frequently diagnosed cancer in U. S. women with >200,000 new cases estimated for 2010. Resistance to standard chemotherapy remains a significant clinical challenge. Thus, the identification of novel molecular biomarkers that predict response to treatment may improve therapeutic decision making and outcomes. Recently it has been reported that the level and activity of the dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and more aggressive primary tumor specimens. Previously, our laboratory has determined that PKR is a key regulator of cell proliferation, apoptosis and transformation. PKR is activated by diverse cellular stresses including growth factor withdrawal from hematopoietic cells, inflammatory cytokine treatment or viral infection to initiate apoptosis presumably as a result eIF2α phosphorylation and inhibition of protein synthesis. However, activated PKR may also promote apoptosis by inhibiting Bcl2 phosphorylation and regulate cell proliferation by mechanisms dependent on p53, STAT1 or NF-κB. In order to gain insight into how PKR signaling may affect breast cancer development and sensitivity to chemotherapy, we measured the level of PKR in primary breast cancer (and paired, normal adjacent tissue) by immunohistochemical staining of a breast tumor tissue microarray containing 50 cases. Increased PKR staining was observed in all tumor types tested (infiltrating ductal carcinoma, mucinous adenocarcinoma, medullary carcinoma, Paget's disease, carcinosarcoma, cystosarcoma phyllodes and benign fibroadenoma) compared to normal adjacent tissue. Furthermore, elevated PKR expression directly correlates with increased tumor grade indicating a more aggressive phenotype. To determine whether PKR may be required for tumor response to doxorubicin, we employed a siRNA strategy to knock-down PKR by >75% in several breast cell lines. Interestingly, basal PKR activity is dramatically higher in those cell lines derived from human mammary carcinoma (i.e. MCF7 and T-47D) compared to a “nontransformed” mammary epithelial cell line (MCF10A). Furthermore, timecourse and dose response studies with doxorubicin, a frequently used and effective chemotherapy, revealed that cells with reduced PKR are significantly less sensitive to the drug compared to control siRNA cells with higher PKR. Interestingly, phosphorylation of eIF2α is unaffected by reduced PKR expression in MCF7 cells even with the highest dose of doxorubicin. This indicates that the mechanism by which PKR promotes doxorubicin sensitivity may be independent of eIF2α phosphorylation and protein synthesis inhibition. Together, these results suggest that increased PKR expression may be a useful biomarker for more aggressive breast tumors and for predicting response to doxorubicin treatment. Based on this model, aggressive tumors that do not display elevated PKR are predicted to be resistant to doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2011-203

Abstract 5325: Localized tumor growth in bone: Quantification of tumor burden, bone destruction, and weight-bearing capability in MDA-MB-231 human mammary carcinoma xenograft-bearing immunodeficient rats

Abstract Metastatic bone disease is common in multiple myeloma, breast, prostate, and lung cancer patients and is a frequent cause of morbidity in advanced disease. Metastatic bone tumors destroy bone and cause severe bone remodeling which leads to structural weakening and bone fractures and often results in severe bone pain in a large percentage of patients. These studies examined tumor growth, bone erosion, and pain in a nude rat model. A surgical defect was created in the tibia, exposing the marrow cavity. Luciferase-expressing MDA-MB-231 mammary tumor cells were injected into the exposed bone cavity. Rats inoculated with tumor cells were treated with either doxorubicin (3 mg/kg/week, iv) or risedronate (80 ug/kg/day, ip). Animals were monitored weekly for tumor burden by optical imaging and for bone erosion by CT. In addition to clinical observations, weight bearing was measured using an incapacitance tester with separate force transducers for each hind paw. Compared to controls, rats treated with doxorubicin demonstrated a significant decease in luminescence, indicating tumor growth inhibition (bone protection). Decreased luminescence correlated with sparing of the bone as measured by CT. In contrast, the bisphosphonate, risedronate, had limited effect on tumor growth, but preserved bone integrity. In both treatments, protection of bone was associated with normalization of weight-bearing. This finding supports the premise that pain in metastatic bone cancer is attributable to bone loss (destruction) and remodeling. Weight-bearing data correlated directly with clinical observations This model, leveraging multiple imaging modalities, enables longitudinal and quantitative assessment of tumor growth; bone loss, remodeling and sparing, and weight-bearing in test animals that provides a sensitive and robust screening tool to evaluate compounds for anti-tumor, bone-sparing and pain amelioration effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5325. doi:10.1158/1538-7445.AM2011-5325

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Latent transforming growth factor binding protein 4 (LTBP4) is downregulated in mouse and human DCIS and mammary carcinomas

BackgroundTransforming growth factor beta (TGF-ß) is able to inhibit the proliferation of epithelial cells and is involved in the carcinogenesis of mammary tumors. Three latent transforming growth factor-ß binding proteins (LTBPs) are known to modulate TGF-ß functions.MethodsThe current study analyses the expression profiles of LTBP4, its isoforms LTBP1 and LTBP3, and TGF-ß1, TGF-ß2, TGF-ß3, and SMAD2, SMAD3 and SMAD4 in human and murine (WAP-TNP8) DCIS compared to invasive mammary tumors. Additionally mammary malignant (MCF7, Hs578T, MDA-MB361) and non malignant cell lines (Hs578BsT) were analysed. Microarray, q-PCR, immunoblot, immunohistochemistry and immunofluorescence were used.ResultsIn comparison to non-malignant tissues (n = 5), LTBP4 was downregulated in all human and mouse DCIS (n = 9) and invasive mammary adenocarcinomas (n = 5) that were investigated. We also found decreased expression of bone morphogenic protein 4 (BMP4) and increased expression of its inhibitor gremlin (GREM1). Treatment of the mammary tumor cell line (Hs578T) with recombinant TGF-ß1 rescued BMP4 and GREM1 expression.ConclusionWe conclude that the lack of LTBP4-mediated targeting in malignant mammary tumor tissues may lead to a possible modification of TGF-ß1 and BMP bioavailability and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-011-0023-y) contains supplementary material, which is available to authorized users.

Simvastatin Prevents Skeletal Metastasis of Breast Cancer by an Antagonistic Interplay between p53 and CD44

Substantial data from clinical trials and epidemiological studies show promising results for use of statins in many cancers, including mammary carcinoma. Breast tumor primarily metastasizes to bone to form osteolytic lesions, causing severe pain and pathological fracture. Here, we report that simvastatin acts as an inhibitor of osteolysis in a mouse model of breast cancer skeletal metastasis of human mammary cancer cell MDA-MB-231, which expresses the mutant p53R280K. Simvastatin and lovastatin attenuated migration and invasion of MDA-MB-231 and BT-20 breast tumor cells in culture. Acquisition of phenotype to express the cancer stem cell marker, CD44, leads to invasive potential of the tumor cells. Interestingly, statins significantly decreased the expression of CD44 protein via a transcriptional mechanism. shRNA-mediated down-regulation of CD44 markedly reduced the migration and invasion of breast cancer cells in culture. We identified that in the MDA-MB-231 cells, simvastatin elevated the levels of mutated p53R280K, which was remarkably active as a transcription factor. shRNA-derived inhibition of mutant p53R280K augmented the expression of CD44, leading to increased migration and invasion. Finally, we demonstrate an inverse correlation between expression of p53 and CD44 in the tumors of mice that received simvastatin. Our results reveal a unique function of statins, which foster enhanced expression of mutant p53R280K to prevent breast cancer cell metastasis to bone.

Retraction: The Chemokine Receptor CXCR6 and Its Ligand CXCL16 Are Expressed in Carcinomas and Inhibit Proliferation

The chemokine receptor CXCR6 and its ligand CXCL16 are involved in inflammation. Thus far, they were known to be expressed mainly by T cells and macrophages, respectively. However, we detected both in all of 170 human primary mammary carcinomas and at similar levels in all 8 human mammary carcinoma cell lines tested by microarray analysis. Expression was confirmed by reverse transcription-PCR and for the cell lines also by fluorescence-activated cell sorting analysis. CXCR6 and CXCL16 were also detected in several mouse and human mammary, colon, and pancreatic carcinoma cell lines. CXCL16 is a transmembrane protein from which the soluble chemokine can be cleaved off. The transmembrane form is present on the surface of the carcinoma cells. Surprisingly, suppression of either CXCR6 or CXCL16 led to greatly enhanced proliferation in vitro as well as in vivo, indicating that their interaction inhibits proliferation. This notion was verified using inhibitory antibodies and by introduction of CXCL16 into a rare CXCL16-negative cell line. The effect was mediated by the G protein-coupled receptor CXCR6 because it was blocked by the G(i) protein inhibitor pertussis toxin. In contrast, the soluble CXCL16 chemokine enhanced proliferation, and this was also mediated by CXCR6 but not via G(i) protein. It is remarkable that both CXCR6 and CXCL16 are expressed by all mammary carcinomas because cells that lose either acquire a growth advantage and should be selected during tumor progression. This suggests an unknown important role in tumor formation. Proteases, possibly macrophage derived, might convert inhibitory transmembrane CXCL16 into the stimulatory chemokine.

Dicofol degradation to p,p′-dichlorobenzophenone – A potential antiandrogen

In the present investigation, the degradation of the acaricide dicofol (also known as kelthane) was investigated with special emphasis on generation of p,p′-dichlorobenzophenone (DCBP) under alkaline conditions as well as induced by UV-light. Dicofol was also incubated in the presence and absence of microsomal preparations to measure potential metabolic formation of DCBP. The results indicate that the degradation of dicofol to DCBP primarily proceeds as an abiotic process via hydroxide ion catalysed elimination of a trichloromethyl anion. The generated anion picks up a proton from the solvent to generate chloroform. Microsomal metabolism does not appear to play a major role in the degradation of dicofol. DCBP is structurally analogous to the antiandrogen p,p′-dichlorodiphenylethene (DDE). We therefore investigated whether DCBP displays antiandrogenic properties. In an in vitro transactivation system utilising transiently transfected African green monkey kidney (COS-7) cells, DCBP showed potent antiandrogenic efficacy. This finding was confirmed by further studies in T47D human mammary carcinoma cells by measuring mRNA and protein expression of androgen dependent genes i.e. TRMP-2 (testosterone-repressed prostate message-2) mRNA and PSA (prostate-specific antigen) protein.

The Effects of n-3 Fatty Acids and Bexarotene on Breast Cancer Cell Progression

Breast cancer cell growth can be inhibited in vivo by retinoid X receptor (RXR) specific retinoids. In both animal and cell culture studies, omega-3 fatty acids share growth regulatory effects similar to those of RXR specific retinoids (rexinoids). One synthetic rexinoid, bexarotene (LCD 1069, Targretin), is used clinically to treat cancer patients. Of concern is that some patients are unable to tolerate high doses of such treatment drugs. We hypothesized that n-3 fatty acids and bexarotene may work synergistically to slow breast cancer cell growth. To test our hypothesis, we used MCF-7 human mammary carcinoma cells and an in vitro cell culture model. We investigated the relationship between the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) alone and in conjunction with bexarotene in slowing MCF-7 cell growth. Following a 72 hr incubation with the respective treatments, bexarotene enhanced cell growth (p < 0.05) while DHA showed a strong growth inhibitory effect which was not enhanced by the addition of bexarotene (p < 0.05). EPA alone was not effective in altering cell growth (p < 0.05). Interestingly, when combined with bexarotene, EPA was more effective at slowing cell growth than when cells received EPA alone. Thus, select omega-3 fatty acids alone are more effective than bexarotene in slowing MCF-7 cell progression. However, the use of the RXR-selective retinoids may enhance the growth regulatory mechanisms of the fatty acid EPA.

AKT Inhibition Relieves Feedback Suppression of Receptor Tyrosine Kinase Expression and Activity

Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.

Abstract P1-06-03: Chinese Herb Fructus Cornii Exhibits Preventive Efficacy in a Cell Culture Model for Hormone Responsive Breast Cancer

Abstract Background: Selective estrogen receptor modulators and combination of chemotherapeutic agents represent conventional therapeutic interventional strategies for estrogen receptor positive (ER+) clinical breast cancer. Herbal medicines have been extensively used in alternative and complementary medicine for cancer of various organ sites. Concerns of adverse toxicity or acquired tumor resistance from conventional chemo-endocrine therapy, rationalizes the use of herbal medicines independently or in combination with chemo-endocrine therapy. The present study has utilized a human cell culture model for ER+ breast cancer to assess potential preventive efficacy of a popular Chinese nutritional herb Fructus Cornii (FC). Material and Methods: The ER+ human mammary carcinoma derived MCF-7 cells represented the model. Status of cellular metabolism of estradiol (E2), anchorage dependent growth and anchorage independent colony formation provided the quantitative end point biomarkers for efficacy. Results: MCF-7 cells maintained in a serum depleted medium (E2&amp;lt;1 nM) retained E2 responsiveness as evidenced by growth modulation by E2, its metabolites, anti-estrogen Tamoxifen, and by the persistence of E2 metabolism via the C17-oxidation, C2-hydroxylation and C16α-hydroxylation pathways. Treatment of E2 stimulated MCF-7 cells with FC produced cytostatic growth arrest and reduction in the number of anchorage independent colonies in a dose dependent manner. Treatment of MCF-7 cells with FC at the maximum cytostatic concentration increased anti-mitogenic 2-hydroxyestrone (2-OHE1), and decreased pro-mitogenic 16α-hydroxyestrone (16α-OHE1) formation. Discussion: These data identify a mechanistic lead for the efficacy of FC and thereby, validate a human cell culture based approach for rapid prioritization of efficacious herbal medicinal products for prevention/therapy of ER+ clinical breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-06-03.

Trefoil Factor 3 Is Oncogenic and Mediates Anti-Estrogen Resistance in Human Mammary Carcinoma

We report herein that trefoil factor 3 (TFF3) is oncogenic and mediates anti-estrogen resistance in human mammary carcinoma. Forced expression of TFF3 in mammary carcinoma cells increased cell proliferation and survival, enhanced anchorage-independent growth, and promoted migration and invasion. Moreover, forced expression of TFF3 increased tumor size in xenograft models. Conversely, depletion of endogenous TFF3 with small interfering RNA (siRNA) decreased the oncogenicity and invasiveness of mammary carcinoma cells. Neutralization of secreted TFF3 by antibody promoted apoptosis, decreased cell growth in vitro, and arrested mammary carcinoma xenograft growth. TFF3 expression was significantly correlated to decreased survival of estrogen receptor (ER)-positive breast cancer patients treated with tamoxifen. Forced expression of TFF3 in mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth, and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. siRNA-mediated depletion or antibody inhibition of TFF3 significantly enhanced the efficacy of antiestrogens. Increased TFF3 expression was observed in tamoxifen-resistant (TAMR) cells and antibody inhibition of TFF3 in TAMR cells improved tamoxifen sensitivity. Functional antagonism of TFF3 therefore warrants consideration as a novel therapeutic strategy for mammary carcinoma.

EGFR tyrosine kinase targeted compounds: synthesis, docking study, and in vitro antitumor activity of some new quinazoline and benzo[d]isothiazole derivatives

The preparation of new quinazoline and benzo[d]isothiazole-based antitumor agents is described. The target compounds fall into three groups including the N-substituted derivatives 2a–d, the substituted amino derivatives 4–6a–d, and the dimeric compounds 7–9a,b. Docking study of the designed compounds into the ATP binding site of epidermal growth factor receptor (EGFR) tyrosine kinase was performed to compare the binding mode of these compounds to the known EGFR inhibitor, lapatinib. All compounds were tested, in vitro, for their activity against human mammary carcinoma cell line (MCF7) in which EGFR is highly expressed. All compounds showed significant growth inhibitory activity. The remarkable activity of the bis quinazoline derivative 8a (IC50 = 0.06 μg/ml; 1.64 nmol/ml) is to be noted.

Annexin A1 attenuates EMT and metastatic potential in breast cancer

Metastasis is the major cause of carcinoma-induced death, but mechanisms involved are poorly understood. Metastasis crucially involves epithelial-to-mesenchymal transition (EMT), causing loss of epithelial polarity. Here we identify Annexin A1 (AnxA1), a protein with important functions in intracellular vesicle trafficking, as an efficient suppressor of EMT and metastasis in breast cancer. AnxA1 levels were strongly reduced in EMT of mammary epithelial cells, in metastatic murine and human cell lines and in metastatic mouse and human carcinomas. RNAi-mediated AnxA1 knockdown cooperated with oncogenic Ras to induce TGFβ-independent EMT and metastasis in non-metastatic cells. Strikingly, forced AnxA1 expression in metastatic mouse and human mammary carcinoma cells reversed EMT and abolished metastasis. AnxA1 knockdown stimulated multiple signalling pathways but only Tyk2/Stat3 and Erk1/2 signalling were essential for EMT.