SPHK1 regulates proliferation and survival responses in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment. Inhibition of SPHK1 was found to attenuate ERK1/2 and AKT signaling and reduce growth of TNBC cells in vitro and in a xenograft SCID mouse model. Moreover, SPHK1 inhibition by siRNA knockdown or treatment with SKI-5C sensitizes TNBCs to chemotherapeutic drugs. Our findings suggest that SPHK1 inhibition, which effectively counteracts oncogenic signaling through ERK1/2 and AKT pathways, is a potentially important anti-tumor strategy in TNBC. A combination of SPHK1 inhibitors with chemotherapeutic agents may be effective against this aggressive subtype of breast cancer.     

Bisindole-PBD regulates breast cancer cell proliferation via SIRT-p53 axis

In a previous study we reported the role of potent bisindole-PBD conjugate as an inclusion in the arsenal of breast cancer therapeutics. In breast cancer cell proliferation, PI3K/AKT/mTOR pathway plays a crucial role by prosurvival mechanism that inhibits programmed cell death. Here, 2 breast cancer cells lines, MCF-7 and MDA-MB-231 were treated with Vorinostat (suberoylanilide hydroxamic acid / SAHA) and bisindole-PBD (5b). We have investigated the effect on PI3K/AKT/mTOR pathway and SIRT expression including epigenetic regulation. There was consistent decrease in the level of PI3K, AKT, mTOR proteins upon treatment of 5b in both MCF-7 and MDA-MB-231 cell lines compared to untreated controls. Treatment with caspase inhibitor (Q-VD-OPH) confirmed that the effect of 5b on PI3K signaling was ahead of apoptosis. Real time PCR and western blot analysis showed profound reduction in the mRNA and protein levels of SIRT1 and SIRT2. Molecular docking studies also supported the interaction of 5b with various amino acids of SIRT2 proteins. Treatment with 5b caused epigenetic changes that include increase of acetylated forms of p53, increase of histone acetylation at p21 promoter as well as decrease in methylation state of p21 gene. Compound 5b thus acts as SIRT inhibitor and cause p53 activation via inhibition of growth factor signaling and activation of p53 dependent apoptotic signaling. This present study focuses bisindole-PBD on epigenetic alteration putting 5b as a promising therapeutic tool in the realm of breast cancer research.     

Measures of cell turnover (proliferation and apoptosis) and their association with survival in breast cancer.

Our objective was to investigate the prognostic significance of cell turnover (apoptosis and proliferation) in breast cancer patients. Apoptosis was microscopically quantitated on histological sections from 791 breast cancer patients with long-term follow-up (median, 16.3 years). Apoptotic counts were also compared with proliferation data (mitotic counts and MIB-1 labeling); apoptosis data derived from terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay; and pathobiological variables, including p53, erbB-2, and estrogen receptor (ER). High apoptotic counts were associated with increased cellular proliferation, ER negativity, immunopositivity of erbB-2 and p53 (P < 0.0001), and shortened disease-specific survival (DSS; P = 0.0009) and disease-free survival (DFS; P = 0.0006). Other factors associated with shortened DFS and DSS by univariate analysis were high tumor grade, nodal metastases, and large tumor size (P < 0.0001 for each). Multivariate analysis of data for all of the patients demonstrated that tumor size, nodal status, ER, histological grade, and erbB-2 showed independent prognostic value. In node-negative patients, tumor size and mitotic rate per 1000 cells independently predicted DFS (P = 0.0055). Tumor grade was the only independent predictor of DSS. For node-positive patients, tumor size, nodal status, ER, and erbB-2 were independent prognostic factors. The number of mitoses per 1000 was independently associated with DFS (P = 0.043) but not with DSS. Apoptosis data did not provide independent prognostic value in any, node-positive or node-negative, breast cancer patients.     

Prolactin and heregulin override DNA damage-induced growth arrest and promote phosphatidylinositol-3 kinase-dependent proliferation in breast cancer cells

Heregulin (HRG), a ligand of ErbB receptor tyrosine kinases, is a potent mitogenic factor for breast cancer cells. Prolactin (PRL) has also been reported to regulate proliferation in breast cancer cells through its receptor, a member of the type I cytokine receptor family. Cytokine receptors are potent mitogens in hematopoietic cells, where they also override DNA damage-induced growth arrest checkpoints through activation of a phosphatidylinositol-3 kinase (PI3K) signaling pathway. In this study, we assessed the effect of gamma-irradiation on the mitogenic activity of HRG and PRL in breast cancer cells. HRG and PRL enhanced the proliferation of non-irradiated breast cancer cell lines in association with their ability to activate PI3K signaling pathways. Both growth factors also overrode irradiation-induced growth arrest in T47D cells, which resulted in decreased viability after irradiation. An inhibitor of PI3K, LY294002, abrogated growth factor-induced proliferation and the activity of cell cycle-dependent kinases in non-irradiated and irradiated cells. Thus, growth factors acting through distinct receptor families share a similar PI3K-dependent ability to promote proliferation and override DNA damage-induced growth arrest in breast cancer cells. These observations also suggest that selective activation of PI3K-dependent signaling can enhance radiosensitivity in breast cancer cells.     

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.     

t-DARPP regulates phosphatidylinositol-3-kinase-dependent cell growth in breast cancer

Background  

Recent reports have shown that t-DARPP (truncated isoform of DARPP-32) can mediate trastuzumab resistance in breast cancer cell models. In this study, we evaluated expression of t-DARPP in human primary breast tumors, and investigated the role of t-DARPP in regulating growth and proliferation in breast cancer cells.     

Musashi1 regulates breast tumor cell proliferation and is a prognostic indicator of poor survival

Background  

Musashi1 (Msi1) is a conserved RNA-binding protein that regulates the Notch and Wnt pathways, and serves as a stem cell marker in the breast and other tissues. It is unknown how Msi1 relates to other breast cancer markers, whether it denotes tumor initiating cells (TICs), and how it affects gene expression and tumor cell survival in breast cancer cells.     

蛋白激酶SGK3过表达对乳腺癌细胞株MDA-MB-231影响的研究

目的 血清和糖皮质激素诱导的蛋白激酶3 (serum and glucocorticoid-inducible protein kinase 3,SGK3)参与细胞增殖、分化和物质转运等生命活动,在某些肿瘤形成中担任重要角色.本研究设计观察SGK3过表达对乳腺癌细胞株MDA-MB-231细胞增殖和存活的影响,并初步探讨其作用的分子机制.方法 免疫组化方法检测乳腺癌组织中SGK3蛋白的表达;通过GenEscortTMⅠ介导用重组质粒pEGFPN1-SGK3瞬时转染乳腺癌MDA-MB-231细胞,荧光显微镜和蛋白质印迹法鉴定转染细胞中融合蛋白SGK3-GFP的表达;细胞增殖实验观察转染细胞的生长增殖情况;流式细胞术分析转染细胞的细胞周期时相变化;蛋白质印迹方法检测细胞增殖与存活相关基因的表达.结果 SGK3在乳腺癌组织中的表达量明显高于癌旁组织和正常乳腺组织(F=29.672,P＜0.001),与二者组间比较,均P＜0.01.荧光显微镜下可见转染细胞因外源基因的表达而发出绿色荧光,且蛋白质印迹法检测可见融合蛋白SGK3-GFP的表达;过表达SGK3的MDA-MB-231细胞,其细胞生长速度加快,细胞倍增时间缩短(F=19.557,P-0.001),与亲本细胞及转染空载体细胞组间比较均P＜0.01.与亲本细胞和转染空载体组比较,SGK3过表达使细胞凋亡比例明显下降(F=12.156,P=0.008),与前两者组间比较P值分别为0.019和0.030;细胞内源性Bad的表达量降低(F=11.152,P=0.010),与前两者组间比较P值分别为0.008和0.005;而Bcl-xL(F=8.810,P=0.016,与前两者组间比较P值分别为0.014和0.009)以及p-GSK3β(F=42.253,P＜0.001,与前两者组间比较P值分别为＜0.001和0.009)的表达量均升高.结论 乳腺癌组织高表达SGK3,SGK3过表达可促进MDA-MB-231细胞的增殖与存活,影响细胞增殖与存活相关基因的表达可能是其发挥作用的主要分子机制.     

SRC-3 is required for prostate cancer cell proliferation and survival

Prostate cancer is the most common cancer in men in America. Currently, steroid receptor coactivators have been proposed to mediate the development and progression of prostate cancer, at times in a steroid-independent manner. Steroid receptor coactivator-3 (SRC-3, p/CIP, AIB1, ACTR, RAC3, and TRAM-1) is a member of the p160 family of coactivators for nuclear hormone receptors including the androgen receptor. SRC-3 is frequently amplified or overexpressed in a number of cancers. However, the role of SRC-3 in cancer cell proliferation and survival is still poorly understood. In this study, we show that SRC-3 is overexpressed in prostate cancer patients and its overexpression correlates with prostate cancer proliferation and is inversely correlated with apoptosis. Consistent with patient data, we have observed that reduction of SRC-3 expression by small interfering RNA decreases proliferation, delays the G1-S transition, and increases cell apoptosis of different prostate cancer cell lines. Furthermore, with decreased SRC-3 expression, proliferating cell nuclear antigen and Bcl-2 expression, as well as bromodeoxyuridine incorporation in prostate cancer cells are reduced. Finally, knockdown of SRC-3 with inducible short hairpin RNA expression in prostate cancer cells decreased tumor growth in nude mice. Taken together, these findings indicate that SRC-3 is an important regulator of prostate cancer proliferation and survival.     

ICAM-3-induced cancer cell proliferation through the PI3K/Akt pathway

ICAM-3 interacts with LFA1, and is involved in the intercellular adhesion of leukocytes as well as in the mainenance of cell survival. It has also been suggested to induce cancer cell proliferation but the precise signaling pathway is unclear. The aim of this study was to determine the ICAM-3-activated downstream pathway in H1299 lung cancer cells. The level of ICAM-3-induced cell growth was examined using BrdU incorporation, which is a colony-forming assay, FACS analysis, and cell counting. The results showed that ICAM-3 expression induces cancer cell proliferation. In addition, FAK, Akt, PDK1, GSK-3beta, BAD, and PTEN were phosphorylated by ICAM-3-overexpression, resulting in enhanced cell proliferation. In conclusion, ICAM-3 expression induces cancer cell proliferation, and an increase in ICAM-3 expression can contribute to cancer progression.     

Intermedin is overexpressed in hepatocellular carcinoma and regulates cell proliferation and survival

Angiogenesis is one of the hallmarks of tumor growth and metastasis. Identification of tumor angiogenic factors has been a critical component in understanding cancer biology and treatment. Intermedin (IMD) has been reported to promote angiogenesis in a rat ischemic model and human umbilical vascular endothelial cells. Our study sought to determine the role of IMD in human hepatocellular carcinoma tumor progression. High IMD mRNA expression levels were observed in human hepatocellular carcinoma tumors, even in early stage disease, by real-time RT-PCR. Immunohistochemical analysis of hepatocellular carcinoma clinical samples demonstrated that the tumor regions were significantly more immunoreactive for IMD than adjacent benign liver. Inhibition of IMD expression using RNA interference reduced cell proliferation in SK-Hep-1 and SNU-398 cells. Blockage of IMD signaling using either an antagonist peptide or a neutralizing antibody inhibited growth in a dose-dependent manner with concomitant induction of apoptosis, causing cleavage of caspase-8 and downregulation of Gli1 and Bcl2. Conversely, addition of IMD active peptide increased the phosphorylation level of extracellular signal-regulated kinase. Thus, IMD might play an important role in cell proliferation and survival of hepatocellular carcinoma. Our data suggests that IMD is a potential biomarker and therapeutic target for hepatocellular carcinoma.     

A novel CYR61-triggered 'CYR61-alphavbeta3 integrin loop' regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway

The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor alphavbeta3 (>200 times). Small peptidomimetic alphavbeta3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of alphavbeta3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, alphavbeta3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of alphavbeta3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of alphavbeta3, similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G2 phase of the cell cycle and the appearance of sub-G1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of alphavbeta3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a alphavbeta3-activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-alphavbeta3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting alphavbeta3 may simultaneously prevent breast cancer angiogenesis, growth, and chemoresistance.     

Effects of fadrozole and leuprorelin acetate on aromatase activity and cell proliferation in a human breast cancer cell line (SK-BR-3)

Background. In recent years, aromatase inhibitors have been used to treat hormone-dependent breast cancer in postmenopausal women. Although gonadotropin-releasing hormone (GnRH) agonists inhibit the growth of breast cancers by estrogen deprivation, it is not known whether GnRH agonists have a direct effect on breast cancer cells. In the present study, we examined the direct effect of a GnRH agonist (leuprorelin acetate) on aromatase activity in a human breast cancer cell line, SK-BR-3. We also studied the synergistic effect of fadrozole (an aromatase inhibitor) and leuprorelin acetate on aromatase activity and cell proliferation in SK-BR-3 cells. Methods. Aromatase activity was determined by measuring [³H] water released upon the conversion of [1β-³H] androstenedione to estrone. Cell proliferation was estimated by determining the incorporation of 5-bromo-2′-deoxyuridine in cellular DNA (cell proliferation assay system). Results. Aromatase activity in SK-BR-3 was inhibited by fadrozole. In addition, SK-BR-3 aromatase activity was inhibited by leuprorelin acetate. Stimulation of cell proliferation by estradiol (10 nM) and testosterone (20 nM) was almost completely inhibited by the addition of an estrogen receptor antagonist, ICI 182780 (10 nM), and fadrozole (1 nM). When both these compounds were added, the most potent inhibition of aromatase activity (fadrozole, 0.1 nM; leuprorelin acetate, 1 nM) and cell proliferation (fadrozole, 10 nM; leuprorelin acetate, 100 nM) was observed. Conclusions. These results lead us to the conclusion that combination therapy with an aromatase inhibitor and a GnRH agonist may provide a new treatment for both pre- and postmenopausal patients with hormone-dependent breast cancer. Received: October 26, 1999 / Accepted: January 26, 2000     

Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1

The ErbB2/3 heterodimer plays a critical role in breast cancer genesis and progression. EBP1, an ErbB3 binding protein, inhibits breast cancer growth but its effects on ErbB3 ligand mediated signal transduction or ErbB receptors is not known. We report here that ectopic expression of EBP1 in MCF-7 and AU565 breast cancer cell lines inhibited HRG-induced proliferation. ErbB2 protein levels were substantially decreased in EBP1 transfectants, while ErbB3 levels were unchanged. HRG-induced AKT activation was attenuated in EBP1 stable transfectants and transfection of a constitutively activated AKT partially restored the growth response to HRG. Down-regulation of EBP1 expression in MCF-7 cells by shRNA resulted in increased cell growth in response to HRG and increased cyclin D1 and ErbB2 expression. These results suggest that EBP1, by down-regulating ErbB signal transduction, attentuates HRG-mediated growth of breast cancer cells.