Claire M. Perks

Insulin-like Growth Factor-binding Protein (IGFBP-3) Predisposes Breast Cancer Cells to Programmed Cell Death in a Non-IGF-dependent Manner

Insulin-like growth factor (IGF) -independent growth inhibition of human breast cancer cells, Hs578T, by IGF-binding protein-3 (IGFBP-3) has previously been demonstrated. Cell growth is a balance between proliferation and programmed cell death (apoptosis). We have investigated whether IGFBP-3 can affect apoptosis of Hs578T cells. As no induction of apoptosis was found, we also investigated its effect on the response to ceramide, an intracellular second messenger that mediates the signal for apoptosis. Using the cell permeable ceramide analogue, C2, induction of apoptosis was established by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, trypan blue uptake, morphological criteria, and flow cytometry. Incubation of cells with non-glycosylated IGFBP-3 (ngIGFBP-3; 0.5–100 ng/ml) resulted in no growth inhibition or increase in apoptosis; whereas, C2 (1–30 μm) resulted in a dose-dependent induction of apoptosis. Addition of IGFs to the cells, alone or with C2, elicited no response in terms of proliferation or survival, respectively. When the cells were preincubated with ngIGFBP-3 before addition of C2 (2–5 μm), apoptosis was accentuated in a dose-dependent manner (at 100 ng/ml IGFBP-3, apoptosis increased from 11 to 88%). In conclusion, we found that IGFBP-3 had no direct inhibitory effect on Hs578T cells but could accentuate apoptosis induced by the physiological trigger ceramide in an IGF-independent manner.

Differential IGF‐independent effects of insulin‐like growth factor binding proteins (1–6) on apoptosis of breast epithelial cells

We have demonstrated previously that insulin‐like growth factor binding protein (IGFBP)‐3 alone has little growth inhibitory effect on Hs578T human breast cancer cells, but that it can dramatically accentuate the apoptotic response to the physiological trigger, ceramide, in an IGF‐independent manner. We have now studied the potential of other IGFBPs (1–6) to interact with apoptotic signalling pathways. Hs578T cells were preincubated with a binding protein (100 ng/ml) for 24 h, followed by co‐incubation of the binding protein with an apoptotic dose of ceramide or RGD‐containing peptide for a further 24 h. Apoptosis was assessed using flow cytometry, MTT (3‐[4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide; thiazolyl blue) assay and morphological assessment. Binding protein profiles were determined using ligand and immunoblotting techniques. Each of the IGFBPs (1–6) alone had no significant (P > 0.05) growth inhibitory effects relative to control cells. In contrast to IGFBP‐3, which significantly (P < 0.05) accentuated C2‐induced apoptosis, IGFBP‐1, ‐2, and ‐6 had no effect, whereas IGFBP‐4 and ‐5 each caused marked (P < 0.01) inhibition of ceramide‐induced programmed cell death. Apoptosis induced by RGD was also significantly (P < 0.05) reduced by IGFBP‐5, whereas IGFBP‐3 had no effect. These data provide evidence to suggest that individual IGFBPs have specific IGF‐independent effects and act differentially on apoptotic signalling pathways. J. Cell. Biochem. 75:652–664, 1999.

Intrinsic actions of IGFBP-3 and IGFBP-5 on Hs578T breast cancer epithelial cells: inhibition or accentuation of attachment and survival is dependent upon the presence of fibronectin

The insulin-like growth factor binding proteins (IGFBPs) have IGF-independent differential effects on cell function. We investigated whether they can affect integrin-receptor-mediated cell attachment to different extracellular matrix (ECM) components in Hs578T cells. Cell attachment to a general ECM gel was unaffected by IGFBP-1 and -6 but was significantly increased by IGFBP-4 and -5 and decreased by IGFBP-2 and -3. Similar results were obtained for attachment to laminin or collagen type IV. Attachment to fibronectin, however, was increased by IGFBP-3 and decreased by IGFBP-5. The actions of IGFBP-3 and -5 on cell attachment to ECM were lost in the presence of a soluble Arg-Gly-Asp (RGD)-containing fibronectin fragment. Thrombospondin reversed the actions of IGFBP-3 on cell attachment, but IGFBP-5 still increased cell attachment. On plastic, neither IGFBP-3 nor -5 alone affected cell viability; although ceramide-induced apoptosis was enhanced by IGFBP-3 but reduced by IGFBP-5. The presence of RGD reversed the action of IGFBP-5 on cell death but attenuated that of IGFBP-3. With cells grown on fibronectin, the action of IGFBP-3 was reversed, and it conferred cell survival, whereas the survival effect of IGFBP-5 was lost. In summary we have demonstrated that IGFBP-3 and -5 both have intrinsic effects on cell survival. In each case the presence of fibronectin or fibronectin fragments determines whether susceptibility to apoptosis is increased or decreased. These effects on cell survival are paralleled by acute effects on integrin receptor function; IGFBP-3 and -5 were able to either enhance or inhibit cell attachment in the presence of fibronectin. Cell survival is tightly controlled by cues from the ECM and from growth factors, particularly the IGFs. Our findings indicate that, in addition to being crucial modulators of IGF actions, the IGFBPs have direct actions on cell attachment and survival that are specific and dependent upon the matrix components present.

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) POTENTIATES PACLITAXEL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

Variability in response to chemotherapy is poorly understood. Paclitaxel‐induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay , cell counting, morphological features and flow cytometry. Pre‐dosing cells with non‐glycosylated insulin‐like growth factor binding protein‐3 (ngIGFBP‐3) had no effect on the cells per se but accentuated paclitaxel‐induced apoptosis. The apoptotic pathway was further examined by measuring caspase‐3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase‐3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP‐3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP‐3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP‐3, which was further increased if the cells had been pre‐dosed with ngIGFBP‐3. These findings suggest that IGFBP‐3 may be an important modulator of paclitaxel‐induced apoptosis. Int. J. Cancer 88:448–453, 2000.

IGFBP-3 PROLONGS THE p53 RESPONSE AND ENHANCES APOPTOSIS FOLLOWING UV IRRADIATION

Neoplastic transformation is characterised by an imbalance in favour of cell growth over programmed cell death (apoptosis). The tumour‐suppressor gene p53, responsible for maintaining cell‐cycle control, is mutated in the majority of human cancers. Loss of function of the target genes of p53 are therefore important in tumourigenesis. One such target gene is the insulin‐like growth factor binding protein‐3 (IGFBP‐3), an extracellular protein responsible for the carriage of IGF‐I but which can act independently of IGF‐I, inhibiting cell growth and enhancing apoptosis. Using the KYSE190 oesophageal carcinoma cell line, we have demonstrated that IGFBP‐3 alone has no effect on cell growth or cell survival. However, it significantly enhanced apoptosis, with a 67% increase in the pre‐G1 peak on flow cytometry following UV irradiation. The increase in p53 was enhanced and prolonged when cells are stressed in the presence of IGFBP‐3. These data suggest an autocrine/paracrine feedback loop exists between IGFBP‐3 and p53, which may provide the social control necessary to maintain normal tissue homeostasis. Int. J. Cancer 88:336–341, 2000.

Differential insulin‐like growth factor (IGF)‐independent interactions of IGF binding protein‐3 and IGF binding protein‐5 on apoptosis in human breast cancer cells. Involvement of the mitochondria.

We have demonstrated previously in Hs578T cells that insulin‐like growth factor binding protein (IGFBP)‐3 can significantly accentuate ceramide (C2)‐induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine‐glycine‐aspartic acid (RGD)‐containing peptide]. In contrast we found that IGFBP‐5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF‐independent actions of IGFBP‐3 and IGFBP‐5 on C2 and RGD‐induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 μmol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 μmol/L) significantly inhibited C2‐induced apoptosis, whereas it significantly accentuated RGD‐induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP‐3 but is not affected by IGFBP‐5. These data indicate that IGFBP‐3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP‐5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria. J. Cell. Biochem. 80:248–258, 2000.

Immunohistochemical expression of insulin-like growth factor binding protein-3 in invasive breast cancers and ductal carcinoma in situ: implications for clinicopathology and patient outcome

IntroductionInsulin-like growth factor binding protein-3 (IGFBP-3) differentially modulates breast epithelial cell growth through insulin-like growth factor (IGF)-dependent and IGF-independent pathways and is a direct (IGF-independent) growth inhibitor as well as a mitogen that potentiates EGF (epidermal growth factor) and interacts with HER-2. Previously, high IGFBP-3 levels in breast cancers have been determined by enzyme-linked immunosorbent assay and immunoradiometric assay methods. In vitro, IGFBP-3s mechanisms of action may involve cell membrane binding and nuclear translocation. To evaluate tumour-specific IGFBP-3 expression and its subcellular localisation, this study examined immunohistochemical IGFBP-3 expression in a series of invasive ductal breast cancers (IDCs) with synchronous ductal carcinomas in situ (DCIS) in relation to clinicopathological variables and patient outcome.MethodsImmunohistochemical expression of IGFBP-3 was evaluated with the sheep polyclonal antiserum (developed in house) with staining performed as described previously.ResultsIGFBP-3 was evaluable in 101 patients with a variable pattern of cytoplasmic expression (positivity of 1+/2+ score) in 85% of invasive and 90% of DCIS components. Strong (2+) IGFBP-3 expression was evident in 32 IDCs and 40 cases of DCIS. A minority of invasive tumours (15%) and DCIS (10%) lacked IGFBP-3 expression. Nuclear IGFBP-3 expression was not detectable in either invasive cancers or DCIS, with a consistent similarity in IGFBP-3 immunoreactivity in IDCs and DCIS. Positive IGFBP-3 expression showed a possible trend in association with increased proliferation (P = 0.096), oestrogen receptor (ER) negativity (P = 0.06) and HER-2 overexpression (P = 0.065) in invasive tumours and a strong association with ER negativity (P = 0.037) in DCIS. Although IGFBP-3 expression was not an independent prognosticator, IGFBP-3-positive breast cancers may have shorter disease-free and overall survivals, although these did not reach statistical significance.ConclusionsIncreased breast epithelial IGFBP-3 expression is a feature of tumorigenesis with cytoplasmic immunoreactivity in the absence of significant nuclear localisation in IDCs and DCIS. There are trends between high levels of IGFBP-3 and poor prognostic features, suggesting that IGFBP-3 is a potential mitogen. IGFBP-3 is not an independent prognosticator for overall survival or disease-free survival, to reflect its dual effects on breast cancer growth regulated by complex pathways in vivo that may relate to its interactions with other growth factors.

A non-IGF binding mutant of IGFBP-3 modulates cell function in breast epithelial cells

We demonstrated previously that IGFBP-3 alone had no effect on cell death, but dramatically modulated apoptosis in Hs578T IGF non-responsive cells. We investigated whether a non-IGF binding mutant of IGFBP-3 retained its intrinsic actions in this cell line, prior to investigating its actions in IGF-responsive cells (MCF-7 and MCF-10A). In the Hs578T cells, the ceramide analogue, C2-induced apoptosis, non-glycosylated, glycosylated or mutant IGFBP-3 alone had no effect but on co-incubation with C2, all forms of IGFBP-3 markedly accentuated triggered apoptosis. In MCF-7 cells, IGFBP-3 was unable to modulate C2-induced death. In the MCF-10A cells, IGFBP-3 acted as a potent survival factor. IGFBP-3 also affected cell growth in the MCF-10A cells (inhibiting at low doses but increasing growth at higher concentrations). These actions of IGFBP-3 in the MCF-10A cells were independent of IGF-1. IGFBP-3 has differential IGF-independent effects on cell death and growth in normal breast and breast cancer cells.

Signalling pathways involved in the direct effects of IGFBP‐5 on breast epithelial cell attachment and survival

We have demonstrated previously that IGFBP‐5 can confer survival against apoptosis induced by ceramide, C2, or a small synthetic arginine–glycine‐aspartic acid (RGD)‐containing peptide in a direct manner. The endogenous ceramide‐induced pathway is normally counter‐balanced by survival signals mediated by sphingosine kinase (SK) and protein kinase C (PKC). In order to investigate whether these pathways are involved in the IGFBP‐5 survival effect, we have used inhibitors of SK (N, N‐di‐methyl sphingosine, DMS) and PKC (chelerythrine chloride, CC). The effect of pre‐incubating Hs578T breast cancer cells with IGFBP‐5 on cell adhesion or on subsequent cell death induced by C2 or RGD was investigated with and without the presence of DMS or CC. Cell death was determined by trypan blue cell counts and apoptosis confirmed by morphological assessment and flow cytometry. Cell attachment was determined by a cell adhesion assay. The presence of IGFBP‐5 significantly inhibited cell death induced by C2 or RGD, compared to the triggers of apoptosis alone (Pu2009<u20090.01 in both cases). In the presence of either IGFBP‐5, CC or DMS, there was no significant effect on cell death compared to the control. IGFBP‐5 in the presence of either inhibitor resulted in a significant increase in cell death; IGFBP‐5 also lost its ability to confer survival on C2 and RGD‐induced apoptosis and in contrast significantly increased cell death. In the cell adhesion assay, IGFBP‐5 significantly increased cell attachment over basal levels. In the presence of either inhibitor the IGFBP‐5 effect on cell adhesion was reversed and cell attachment was reduced to below basal levels. These data suggest that IGFBP‐5 promotes the attachment and survival of Hs578T cells by modulating the balance between ceramide and opposing survival signals. J. Cell. Biochem. 84: 784–794, 2002.

Changes in the levels of integrin and focal adhesion kinase (FAK) in human melanoma cells following 532 nm laser treatment

Despite the increase in laser therapy, concern remains that sublethal treatment of pre‐malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the β1 integrin in laser vs. non‐laser treated cells. We treated cells with laser generated from a frequency doubled Q‐switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with β1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4‐ and 11.2‐fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both α3 and 4 integrins were up‐regulated, by approximately 4‐fold and 7‐ to 9‐fold, respectively. In addition, the β1 integrin was proteolysed in both cell lines and the levels of FAK associated with β1 was increased (2.1‐ and 2.7‐fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology. Int. J. Cancer 82:353–358, 1999.