Simultaneous inhibition of focal adhesion kinase and SRC enhances detachment and apoptosis in colon cancer cell lines

Focal adhesion kinase (FAK) and Src have been shown to be overexpressed in colon cancer. We have studied the role of these two kinases in resistance to apoptosis. Adenovirus-containing FAK-CD (Ad-FAK-CD), a dominant-negative, COOH-terminal portion of FAK, was used to inhibit FAK and cause apoptosis. Colon cancer cell lines were more resistant to Ad-FAK-CD-induced detachment and apoptosis than the breast cancer cell line, BT474. Colon cancer cell lines overexpressed highly active Src and FAK. Ad-FAK-CD-induced apoptosis was significantly increased by PP2, an inhibitor of Src family kinases. Activation of caspase-3, down-regulation of FAK, and Src and AKT activities were demonstrated in Ad-FAK-CD + PP2-treated colon cancer cells undergoing apoptosis. The results suggest that FAK and Src are both important survival factors, playing a role in protecting colon cancer cell lines from Ad-FAK-CD-induced apoptosis. Dual inhibition of these kinases may be important for therapies designed to enhance the apoptosis in colon cancers.     

The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.     

HER2 signaling downregulation by trastuzumab and suppression of the PI3K/Akt pathway: an unexpected effect on TRAIL-induced apoptosis

We investigated whether HER2 downregulation by trastuzumab modulates the responsiveness of breast cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Interestingly, in contrast to increased response to TRAIL in SKBr3 cells, trastuzumab decreased the susceptibility of BT474 cells to TRAIL. This decrease was also observed after exogenous inhibition of PI3-K/Akt kinase, but not MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK). In BT474 cells, but not SKBr3 cells, inhibition of the HER2/phosphatidylinositol 3' kinase (PI3K)/Akt pathway resulted in downregulation of the pro-apoptotic receptors TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2. TRAIL-induced caspase-8 activation, Bid processing, drop of DeltaPsi(m), and poly ADP-ribose polymerase (PARP) cleavage but not in caspase-9 activation, and these events were inhibited in HER2/PI3K/Akt-suppressed BT474 cells, which on the other hand exhibited downregulation of Bcl-xL and increased response to mitomycin C. We show that HER2/PI3K/Akt pathway may play a specific pro-apoptotic role in certain cell type by inducing TRAIL-R1 and -R2 expression and thereby enhancing responsiveness to TRAIL.     

S100A14, a Member of the EF-hand Calcium-binding Proteins,Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.     

Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.     

Overexpression of profilin reduces the migration of invasive breast cancer cells

The exact role profilin plays in cell migration is not clear. In this study, we have evaluated the effect of overexpression of profilin on the migration of breast cancer cells. Overexpression was carried out by stably expressing GFP-profilin in BT474 cells. It was observed that even a moderate level of overexpression of profilin significantly impaired the ability of BT474 cells to spread on fibronectin-coated substrate and migrate in response to EGF. GFP-profilin expressing cells also showed increased resistance to detachment in response to trypsin and increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin compared to the parental and GFP-expressing (control) cell lines. These results suggest that perturbation of profilin levels may offer a good strategy for controlling the metastatic potential of breast cancer cells.     

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.     

Knockdown of ribosomal protein S15A inhibits proliferation of breast cancer cells through induction of apoptosis in vitro

To explore the role of ribosomal protein S15A (RPS15A) in breast cancer. The Oncomine database was used to compare the expression of RPS15A in human breast cancer tissues and normal tissues. RPS15A in breast cancer cell line ZR-75-30 and BT474 was specifically knocked down using lentivirus-mediated short hairpin RNAs (shRNAs). RPS15A knockdown efficiency was validated by quantitative polymerase chain reaction and western blot analysis. Subsequently, the functional effects of RPS15A on proliferation of breast cancer cells were investigated by MTT, colony formation and flow cytometry assays. Functional analysis indicated that RPS15A knockdown could inhibit cell proliferation, induced cell cycle arrest and apoptosis. Mechanism analysis revealed RPS15A mediated apoptosis via activating of caspase-3 and PARP cleavage, upregulating of Bad and BAX and downregulating of Bcl-2. Our preliminary study highlighted the importance of RPS15A in breast cancer growth. The inhibition of RPS15A may be a promising therapeutic target for breast cancer treatment.     

Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation

OBJECTIVES: The potential of epidermal growth factor receptor (EGFR)- and Her2-targeted antibodies Cetuximab, Pertuzumab and Trastuzumab, used in combination to inhibit cell proliferation of breast cancer cells in vitro, has not been extensively investigated. It is anticipated that there would be differences between specific erbB receptor co-expression profiles that would affect tumour cell growth. MATERIALS AND METHODS: We have examined the effects of Cetuximab, Pertuzumab and Trastuzumab, applied separately or in combination, on cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. Cell cycle progression of BT474 and SK-BR-3 cells was statically and dynamically assessed using flow cytometry. In order to discover a potential influence of differential EGFR co-expression on sensitivity to antibody treatment, EGFR was down-regulated by siRNA in SK-BR-3. An annexinV/propidium iodide assay was used to identify potential induction of apoptosis. RESULTS: Treatment with Pertuzumab and Trastuzumab, both targeted to Her2, resulted in a reduced fraction of proliferating cells, prolongation of G(1) phase and a great increase in quiescent BT474 cells. Cetuximab had no additional contribution to the effect of either Pertuzumab or Trastuzumab when administered simultaneously. Treatment with the antibodies did not induce an appreciable amount of apoptosis in either BT474 or SK-BR-3 cells. In contrast to SK-BR-3, the BT474 cell line appears to be more sensitive to antibody treatment due to low EGFR content besides Her2 overexpression. CONCLUSION: The extent of decelerated or blocked cell proliferation after antibody treatment that is targeted to EGFR and to Her2 depends both on EGFR and Her2 co-expression and on antibody combination used in the treatment setting. Cetuximab did not enhance any inhibitory effect of Trastuzumab or Pertuzumab, most probably due to the dominant overexpression of Her2. Cell susceptibility to Trastuzumab/Pertuzumab, both targeted to Her2, was defined by the ratio of EGFR/Her2 co-expression.     

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa^®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.     

Disintegrin causes proteolysis of beta-catenin and apoptosis of endothelial cells. Involvement of cell-cell and cell-ECM interactions in regulating cell viability

Disintegrins, the snake venom-derived arginine-glycine-aspartic acid-containing peptides, have been demonstrated to inhibit angiogenesis through induction of endothelial cell apoptosis. However, it is not clear how a disintegrin causes endothelial apoptosis. In this study, we elucidated the action mechanism of disintegrin in causing endothelial apoptosis by using rhodostomin as a tool. We showed that cell detachment was observed at the early stage of rhodostomin treatment. It was initiated through the blockade by integrin alphanubeta3 and was accelerated by a mechanical stretch from neighboring cells. Both rhodostomin and poly(HEME) induced a higher percentage of cells at G2-M phase, the cleavage of beta-catenin and poly(ADP-ribose) polymerase during apoptosis, indicating that cell detachment is a prerequisite for rhodostomin-induced apoptosis. Moreover, pp125(FAK) phosphorylation and actin cytoskeleton were affected upon rhodostomin treatment. The activation of caspase-3 but not that of caspase-9 was detected after rhodostomin treatment. In addition, general caspase inhibitors inhibited the cleavage of beta-catenin and poly(ADP-ribose) polymerase, and DNA fragmentation, whereas they did not prevent cell shape change or detachment. According to these results, we concluded that disintegrin-induced endothelial apoptosis is a complex process, not merely caused by a blockade of endothelial integrin alphanubeta3 but also by an accompanied shape change and mechanical stretches among cells.     

SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.     

Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model

Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 μmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.     

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

BACKGROUND: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. METHODS: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. RESULTS: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. CONCLUSION: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.     

Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation

Epidermal growth factor (EGF) stimulates the homodimerization of EGF receptor (EGFR) and the heterodimerization of EGFR and ErbB2. The EGFR homodimers are quickly endocytosed after EGF stimulation as a means of down-regulation. However, the results from experiments on the ability of ErbB2 to undergo ligand-induced endocytosis are very controversial. It is unclear how the EGFR-ErbB2 heterodimers might behave. In this research, we showed by subcellular fractionation, immunoprecipitation, Western blotting, indirect immunofluorescence, and microinjection that, in the four breast cancer cell lines MDA453, SKBR3, BT474, and BT20, the EGFR-ErbB2 heterodimerization levels were positively correlated with the ratio of ErbB2/EGFR expression levels. ErbB2 was not endocytosed in response to EGF stimulation. Moreover, in MDA453, SKBR3, and BT474 cells, which have very high levels of EGFR-ErbB2 heterodimerization, EGF-induced EGFR endocytosis was greatly inhibited compared with that in BT20 cells, which have a very low level of EGFR-ErbB2 heterodimerization. Microinjection of an ErbB2 expression plasmid into BT20 cells significantly inhibited EGF-stimulated EGFR endocytosis. Coexpression of ErbB2 with EGFR in 293T cells also significantly inhibited EGF-stimulated EGFR endocytosis. EGF did not stimulate the endocytosis of ectopically expressed ErbB2 in BT20 and 293T cells. These results indicate that ErbB2 and the EGFR-ErbB2 heterodimers are impaired in EGF-induced endocytosis. Moreover, when expressed in BT20 cells by microinjection, a chimeric receptor composed of the ErbB2 extracellular domain and the EGFR intracellular domain underwent normal endocytosis in response to EGF, and this chimera did not block EGF-induced EGFR endocytosis. Thus, the endocytosis deficiency of ErbB2 is due to the sequence of its intracellular domain.