alpha-Tocopheryl succinate-induced apoptosis in human gastric cancer cells is modulated by ERK1/2 and c-Jun N-terminal kinase in a biphasic manner

Gastric neoplastic disease is one of the most frequent causes of cancer-associated deaths with poor prognosis. Here we studied the effect of the redox-silent analogue alpha-tocopheryl succinate (alpha-TOS), a strong apoptogen and anti-cancer agent, on the gastric cancer cell line SGC-7901. alpha-TOS inhibited proliferation of the cells and induced their apoptosis in a concentration- and time-dependent manner, while succinate or alpha-tocopherol showed no effect. The effect of alpha-TOS was modulated by components of the MAPK signaling network, including ERK1/2 and c-Jun N-terminal kinase (JNK), but not p38. Activation of ERK1/2 occurred early and increased until 12h, coinciding with an in crease in apoptosis in the cells, after which it dropped abruptly, while activation of JNK rose steadily, reaching a plateau at 12h of alpha-TOS treatment. The effects of ERK1/2 and JNK on the apoptosis outcome are transmitted via c-Jun, since transfection of the cells with c-Jun antisense oligodeoxynucleotide inhibited alpha-TOS-induced apoptosis. We conclude that ERK1/2 and JNK positively regulate apoptosis induced in gastric cancer cells by alpha-TOS.     

Alpha-tocopheryl succinate alters cell cycle distribution sensitising human osteosarcoma cells to methotrexate-induced apoptosis

alpha-Tocopheryl succinate (alpha-TOS) exerts pleiotrophic responses in malignant cells leading to cell cycle arrest, differentiation and apoptosis. We tested the ability of alpha-TOS to induce apoptosis or cell cycle perturbation in three human osteosarcoma (OS) cell lines which differ in their pRB and p53 status. We found high levels of apoptosis in OS cells carrying wild-type p53 gene when exposed to alpha-TOS, while the mutant p53 cells were resistant. A S/G2 transition arrest was observed in two OS cell lines exposed to alpha-TOS, which sensitised them to methotrexate, an agent whose activity is cell cycle-dependent. We propose that alpha-TOS may be used as a drug or an adjuvant for treatment of osteosarcomas.     

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.     

Activation of the phosphatidylinositol 3-kinase/Akt pathway prevents radiation-induced apoptosis in breast cancer cells

Radiotherapy is widely used in the treatment of breast cancer and reduces the risk of loco-regional recurrence. Overexpression of the erbB2 receptor occurs in 20-30% of all breast cancers, and seems to be involved in chemotherapeutic resistance of breast cancer cells and radioresistance of lung cancer cells. The hypothesis of this study was that erbB2 confers resistance to radiation-induced apoptosis in breast cancer cells through the phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway. Two human breast cancer cell lines were used, BT-474 and MCF-7. BT-474 cells overexpress erbB2 and have mutated p53, while MCF-7 have normal expression of erbB2 and functional p53. The cells were treated with the PI3-K inhibitor wortmannin or the erbB receptor ligand heregulin-beta1, which is expressed by both malignant and stromal cells in vivo. After pharmacological treatment, the cells were irradiated with 10 Gy gamma-radiation. Consistent with the p53 status in the cell lines, gamma-radiation caused G1 arrest in MCF-7 cells, but not in BT-474 cells. 10 Gy gamma-radiation increased apoptosis by on an average 76% (95% CI, 44-109%) in MCF-7. Treatment of MCF-7 with heregulin-beta1 decreased apoptosis by 66% (95% CI, 48-84%) compared to the untreated controls. In BT-474 cells, wortmannin in combination with radiation resulted in 119% (95% CI, 76-161%) more apoptosis compared to wortmannin alone, whereas radiation alone resulted in 45% (95% CI, 15-75%) increased apoptosis. This radiosensitising effect was not seen in MCF-7. Furthermore, transfection of MCF-7 cells with constitutively active Akt made the cells more resistant against apoptosis. Taken together, our results support the hypothesis that the erbB2/PI3-K/Akt signalling pathway is involved in resistance to radiation-induced apoptosis in breast cancer cells in which this signalling pathway is overstimulated.     

RNAi‐mediated downregulation of uPAR synergizes with targeting of HER2 through the ERK pathway in breast cancer cells

Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB‐2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2‐amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.     

Alpha-tocopheryl succinate sensitises a T lymphoma cell line to TRAIL-induced apoptosis by suppressing NF-kappaB activation

Activation of nuclear factor-kappaB (NF-kappaB) can interfere with induction of apoptosis triggered by the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL; Apo2L). Therefore, agents that suppress NF-kappaB activation may sensitise cells to TRAIL-dependent apoptosis. Exposure of Jurkat cells to TRAIL resulted in massive and saturable apoptosis induction, following an initial lag time. This lag was abolished by pretreatment of the cells with subapoptotic doses of alpha-tocopheryl succinate (alpha-TOS) or the proteasome inhibitor MG132. Exposure of the cells to TRAIL led to a rapid, transient activation of NF-kappaB, a process that was suppressed by cell pretreatment with alpha-TOS or MG132. Activation of NF-kappaB by TNF-alpha prior to TRAIL exposure increased resistance of the cells to TRAIL-mediated apoptosis. We conclude that alpha-TOS sensitises cells to TRAIL killing, at least in some cases, through inhibition of NF-kappaB activation. This further supports the possibility that this semisynthetic analogue of vitamin E is a potential adjuvant in cancer treatment, such as in the case of TRAIL-mediated inhibition of cancer.     

HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance

Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumab-induced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2.     

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.     

Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer

Introduction

Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer.

Methods

Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel’s antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo.

Results

MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3.

Conclusions

The combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel.     

Increased Constitutive Activity of PKB/Akt in Tamoxifen Resistant Breast Cancer MCF-7 Cells

The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3 alpha / beta and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGF alpha (transforming growth factor- alpha ) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib ("Iressa"/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.     

Vitamin E succinate induces ceramide-mediated apoptosis in head and neck squamous cell carcinoma in vitro and in vivo.

PURPOSE: Vitamin E succinate (alpha-TOS) inhibits the growth of cancer cells without unacceptable side effects. Therefore, the mechanisms associated with the anticancer action of alpha-TOS, including ceramide-mediated apoptosis, were investigated using head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. EXPERIMENTAL DESIGN: Five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) were treated with alpha-TOS, and its effects on cell proliferation, cell cycle progression, ceramide-mediated apoptosis, and ceramide metabolism were evaluated. The anticancer effect of alpha-TOS was also examined on JHU-022 solid tumor xenograft growth in immunodeficient mice. RESULTS: Alpha-TOS inhibited the growth of all the HNSCC cell lines in vitro in a dose- and time-dependent manner. Thus, JHU-013 and JHU-022 cell lines were more sensitive to alpha-TOS than the other cell lines. Cellular levels of ceramide, sphingomyelinase activity, caspase-3, and p53 were elevated with increasing time of exposure to alpha-TOS. The degradation of poly(ADP-ribose) polymerase protein in JHU-022 cells treated with alpha-TOS provided evidence for apoptosis. The amounts of nuclear factor kappaB, Bcl-2, and Bcl-X(L) proteins were reduced in the cells treated with alpha-TOS for 6 hours. The levels of caspase-9, murine double minute-2, and IkappaB-alpha proteins were unchanged after alpha-TOS treatment. I.p. administration of alpha-TOS slowed tumor growth in immunodeficient mice. CONCLUSIONS: Alpha-TOS showed promising anticancer effects to inhibit HNSCC growth and viability in vivo and in vitro. The induction of enzymes involved in ceramide metabolism by alpha-TOS suggests that ceramide-mediated apoptosis may expand therapeutic strategies in the treatment of carcinoma.     

Hepatotoxicity in cancer patients receiving erb-38, a recombinant immunotoxin that targets the erbB2 receptor.

To exploit overexpression of erbB2 in human cancers, we constructed a single-chain immunotoxin (erb-38) that contains the Fv portion of monoclonal antibody e23 fused to a truncated form of Pseudomonas exotoxin A. In a Phase I study, five breast cancer patients and one esophageal cancer patient received three doses of erb-38 at 1.0 and 2.0 microg/kg. Hepatotoxicity was observed in all patients. Immunohistochemistry showed the presence of erbB2 on hepatocytes explaining the liver toxicity of erb-38. We suggest that targeting of tumors with antibodies to erbB2 armed with radioisotopes or other toxic agents may result in unexpected organ toxicities due to erbB2 on normal cells.     

Activation of fibronectin/PI-3K/Akt2 leads to chemoresistance to docetaxel by regulating survivin protein expression in ovarian and breast cancer cells

The purpose of this study was to investigate the possible role of PI-3K/Akt2 pathway in docetaxel-induced apoptosis. Here we showed that transfection of full-length Akt2 into breast and ovarian cancer cells could provoke Akt phosphorylation and induce an enhanced resistance to docetaxel. FN adhesion promoted Akt phosphorylation in highly metastatic cancer cells A2780 and MDAMB231, and further brought on significant protection for tumor cells against docetaxel-induced apoptosis. Inhibition of Akt2 activity by co-transfection with two shRNA vectors targeting the same Akt2 mRNA or simply by administration with PI 3-Kinase inhibitor Ly294002 counteracted the ability of FN to protect cells from undergoing apoptosis induced by docetaxel. We further showed that Akt2 activation protected against docetaxel-induced apoptosis by regulating survivin levels in a PI 3-Kinase-dependent manner. We conclude that FN/PI-3K/Akt2 pathway might play an important role in inducing resistance to docetaxel in breast and ovarian cancer cells. Our results therefore indicate that the activation of Akt2, promoted by FN attachment, might be critical in determining whether cells survive or undergo apoptosis. Targeting the PI-3K/Akt2 pathway might be a promising strategy for enhancing sensitivity to docetaxel in breast or ovarian cancer.     

Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo.

Alpha-tocopheryl succinate (alpha-TOS), a redox-inactive analogue of vitamin E, is a strong inducer of apoptosis, whereas alpha-tocopherol (alpha-TOH) lacks apoptogenic activity (J. Neuzil et al., FASEB J., 15: 403-415, 2001). Here we investigated the possible antineoplastic activities of alpha-TOH and alpha-TOS and further explored the potential of alpha-TOS as an antitumor agent. Using nude mice with colon cancer xenografts, we found that alpha-TOH exerted modest antitumor activity and acted by inhibiting tumor cell proliferation. In contrast, alpha-TOS showed a more profound antitumor effect, at both the level of inhibition of proliferation and induction of tumor cell apoptosis. alpha-TOS was nontoxic to normal cells and tissues, triggered apoptosis in p53(-/-) and p21(Waf1/Cip1(-/-)) cancer cells, and exerted a cooperative proapoptotic activity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) due to differences in proapoptotic signaling. Finally, alpha-TOS cooperated with tumor necrosis factor-related apoptosis-inducing ligand in suppression of tumor growth in vivo. Vitamin E succinate is thus a potent and highly specific anticancer agent and/or adjuvant of considerable therapeutic potential.     

乳腺癌雌孕激素水平及其受体和c-erbB-2癌基因表达的临床意义

目的 :研究乳腺癌雌孕激素水平、雌孕激素受体及c erbB 2癌基因蛋白的表达情况及其临床意义。方法 :分别应用12 5I放射免疫组化ABC法研究了 82例乳腺癌和 6 8例乳腺良性病变患者血清雌孕激素水平以及病变组织雌孕激素受体、癌基因c erbB 2的表达。结果 :乳腺癌组的雌激素水平和癌基因c erbB 2的表达率明显高于良性病变组 ,雌激素水平与腋淋巴结状况呈明显正相关。ER、PR的表达与腋淋巴结状况关系不大 ,但与临床分期呈明显负相关 ,ER的阳性表达者 5年无瘤生存率明显高于复发或死亡者。癌基因c erbB 2的阳性表达率与临床分期及远期生存率呈显著正相关。结论 :雌孕激素水平高、癌基因c erbB 2的阳性表达是乳腺癌预后不良的指标 ,ER、PR的阳性表达是乳腺癌预后良好的指标 ,对乳腺癌的诊断治疗和预后判断具有重要的指导意义。     

Estrogen suppression of erbB2 expression is associated with increased growth rate of ZR-75-1 human breast cancer cells in vitro and in nude mice.

Amplification and enhanced expression of the erbB2/HER-2/neu gene has been associated with an increased growth rate and poor prognosis of human breast cancer. We have studied the relationship between erbB2 expression and the regulation of cell growth by estrogen and anti-estrogens in the human breast cancer cell line ZR-75-1 in vitro and in athymic nude mice, pS2 being used as a marker gene for estrogen-stimulated gene expression. Only low amounts of erbB2 mRNA were seen in the cells grown in vitro in the presence of estrogen which stimulated the cells to proliferate rapidly and induced the expression of pS2 mRNA. Upon hormone withdrawal, erbB2 mRNA and protein increased, while pS2 mRNA declined to an undetectable level and cell proliferation slowed down. Opposite but more rapid changes were observed upon estrogen addition. The anti-estrogens toremifene and tamoxifen inhibited estrogen induction of pS2 expression, down-regulation of erbB2 expression and proliferation of the ZR-75-I cells in a concentration-dependent manner. Similar results were obtained in nude mice. ZR-75-I cells formed tumors only in mice carrying estrogen pellets. In these tumors little erbB2 mRNA was seen. Concomitant administration of toremifene or tamoxifen increased erbB2 mRNA and abolished pS2 mRNA. Our results show that enhanced expression of erbB2 is associated with hormone deprivation and growth arrest of the estrogen-dependent breast cancer cell line ZR-75-I. Thus, in mammary epithelial cells, erbB2 may have important estrogen-regulated functions which are not related to cell proliferation.     

ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients

Occult hematogenous micrometastases are the major cause for metastatic relapse and cancer-related death in patients with operable primary breast cancer. Although sensitive immunocytochemical and molecular methods allow detection of individual breast cancer cells in bone marrow (BM), a major site of metastatic relapse, current detection techniques cannot discriminate between nonviable shed tumor cells and seminal metastatic cells. To address this problem, we analyzed the relevance of erbB2 overexpression on disseminated cytokeratin-18-positive breast cancer cells in the BM of 52 patients with locoregionally restricted primary breast cancer using immunocytochemical double labeling with monoclonal antibody 9G6 to the p185erbB2 oncoprotein. Expression of p185erbB2 on BM micrometastases was detected in 31 of 52 (60%) patients independent of established risk factors such as lymph node involvement, primary tumor size, differentiation grade, or expression of p185erbB2 on primary tumor cells. After a median follow-up of 64 months, patients with p185erbB2-positive BM micrometastases had developed fatal metastatic relapses more frequently than patients with p185erbB2-negative micrometastases (21 versus 7 events; P = 0.032). In multivariate analysis, the presence of p185erbB2-positive micrometastases was an independent prognostic factor with a hazard ratio of 2.78 (95% confidence interval, 1.11-6.96) for overall survival (P = 0.029). We therefore conclude that erbB2 overexpression characterizes a clinically relevant subset of breast cancer micrometastases.     

The role of erbB2 signal transduction pathways in human breast cancer

Summary The erbB2 receptor is expressed at very high levels in nearly 30% of human breast cancer patients and plays an important role in the transformation and the prognosis of breast cancer. While evidence accumulates to support the relationship between erbB2 overexpression and poor overall survival in human breast cancer, understanding of the biological consequence(s) of erbB2 overexpression remains elusive. Our recent discovery, cloning, sequencing, and expression of the erbB2 ligand (gp30) has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance, and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. Studiesin vitro have shown that gp30 induces a biphasic growth effect (induction of growth at low concentrations and inhibition of growth at high concentrations) on cells with erbB2 over-expression. Strikingly, we have recently observed that the erbB2 signalling pathway can be modulated by estrogen acting through the estrogen receptor (ER). Conversely, we observed that down regulation of erbB2 by estrogen can be blocked by gp30 acting through the erbB2 receptor. Clearly, mechanistic aspects of the erbB2/ligand interaction need to be understood from a therapeutic standpoint, and may furthermore provide additional insights into treatment synergy for particular patients. We think that these studies will facilitate the emergence of erbB2-targeted therapy.     

Harmine suppresses breast cancer cell migration and invasion by regulating TAZ-mediated epithelial-mesenchymal transition

Breast cancer is a highly lethal disease due to cancer metastasis. Harmine (HM), a β-carboline alkaloid, is present in various medicinal plants. Our previous study demonstrated that HM suppresses cell proliferation and migration by regulating TAZ in breast cancer cells and accelerates apoptosis. Epithelial-mesenchymal transition (EMT) plays an important role in the development of breast cancer by inducing the characteristics of cancer stem cells, cancer metastasis and recurrence. Overexpression of TAZ was shown to mediate EMT in breast cancer cells. We aimed to investigate whether HM inhibits EMT and metastasis of breast cancer cells by targeting TAZ. In this study, the cells treated with HM or with downregulated expression of TAZ showed an increase in epithelial markers and decrease in mesenchymal markers in breast cancer cells. Consistently, the breast cancer cells treated with HM or with downregulated expression of TAZ showed suppressed migration and proliferation. Moreover, TAZ overexpression reversed EMT and metastasis induced by HM in breast cancer cells. Thus, HM suppresses EMT and metastasis and invasion by targeting TAZ in breast cancer cells. HM can be used as an anticancer drug for breast cancer treatment and chemoprevention.     

Overexpression of p185 is not related to erbB2 amplification in ovarian cancer

BACKGROUND:: While in breast cancer the amplification and overexpressionof the erbB2 gene has been reported in numerous studies andfound to be correlated to poor prognosis, information aboutthis oncogene with respect to ovarian cancer is still limited.A recent study reported that approximately 30% of tumor biopsiesfrom ovarian cancer patients exhibited erbB2 amplification andoverexpression and suggested that the overexpression of thisoncogene is an indicator of bad prognosis in ovarian cancer.The purpose of our studies was to investigate amplificationof the erbB2 gene, the levels of erbB2 m-RNA and the erbB2 product(pi85) in ovarian cancer, and the correlation between thesefindings and the pathological and clinical features. RESULTS:: Amplification of the erbB2 gene was investigated by Southernblot analysis in 75 samples from 62 patients; mRNA levels wereevaluated by Northern blot analysis in 58 samples from 48 patients;and pl85 was determined by immunohistochemistry in 65 samplesfrom 65 patients. The erbB2 gene was amplified in only one case(1.6%), and a marked Increase in erbB2 mRNA was found only inthe same case. Staining for pl85 was positive in 12 cases (18.5%).The staining was always confined to the cytoplasm except inthe case that showed amplification of erbB2 in which pl85 waslocalized in the membrane. No correlation was found betweenpl85 positivity and pathological and clinical features or responseto chemotherapy. Western blot analysis showed that the molecularweight of pl85 in positive ovarian cancer cells was approximately10 KDa lower than in breast cancer. CONCLUSION:: In contrast to breast cancer, in ovarian cancer the amplificationof erbB2 appears infrequent. The levels of pl85 detected byimmunohistochemistry were not related to the amplification ofthe gene or to the increase in mRNA, suggesting the possibilityof a longer half-life of the protein in the positive cases.The finding that erbB2 product in ovarian cancer is mostly localisedin cytoplasm and not in the membrane as in breast cancer andthat it has a lower molecular weight than the pl85 in breastcancer suggest that this oncogene plays a different biologicalrole in these two neoplasms. oncogene, erbB2, ovarian cancer, amplification