Phosphoinositide 3-kinase targeting by the β galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death

Abstract

IntroductionPhosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls.MethodsAggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells.ResultsIn aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the βGBP cytokine and loss of akt mRNA results in apoptotic death. A functional correlation between ERK and the kt gene was also found. The relationship between ERK, akt mRNA, PI3K and cell vulnerability to βGBP challenge was sustained both in mammary ductal cells forced to mimic an aggressive behaviour and in non-aggressive breast cancer cells undergoing an enforced shift into an aggressive phenotype.ConclusionsβGBP, a newly discovered physiological inhibitor of PI3K, is a selective and potent inducer of apoptosis in aggressive breast cancer cells. Due to its physiological nature, which carries no chemotherapeutic disadvantages, βGBP has the potential to be safely tested in clinical trials.

Responsiveness to PI3K and MEK Inhibitors in Breast Cancer. Use of a 3D Culture System to Study Pathways Related to Hormone Independence in Mice

“…This effect is in accordance with a study that shows that treatment of aggressive breast cancer cells with β galactoside binding protein (βGBP) cytokine, another functional inhibitor of PI3K, induces apoptosis through a reduction of AKT mRNA levels [50]. Furthermore, our results indicate that LY294002 causes inhibition of tumor growth (Figure 1) and increase in lumen formation in C4-HI cancer cells through an intrinsic BAX/mitochondrial/activated caspase-9 apoptotic mechanism (Figure 5).…”

“…Furthermore, inhibition of the two pathways by targeting MEK and PI3K produced synergistic effects in inhibiting cell survival (Figure 4), highlighting the interconnectivity of oncogenic signal transduction circuits. The correlation between ERK and PI3K/AKT signaling has been reported in breast cancer cells [29], [49], [50]. Furthermore, Weigelt et al [28] state that during the acquisition of resistance to targeted therapies, breast cancer cells are able to rapidly adapt to different environments and signaling cues by switching between alternative pathways, specifically PI3K/AKT and RAS-MEK-ERK, that in turn regulate proliferation and cell survival.…”

Solasodine inhibits human colorectal cancer cells through suppression of the AKT/glycogen synthase kinase‐3β/β‐catenin pathway

“…Protein kinase B is important for the anticancer effects of solasodine and our results have verified the suppression of its activation as a critical event when inhibiting CRC cells. At the transcriptional level, solasodine might decrease the stability of AKT mRNA and activate its degradation mechanism through regulation of several molecules, including RNA helicase of the DEAD box family, histone deacetylase, and the β‐galactoside binding protein cytokine . At the translation level, PI3K is one of the primary upstream molecules of AKT, which is a heterodimeric enzyme consisted of p110 catalytic and p85 regulatory subunits.…”

“…At the transcriptional level, solasodine might decrease the stability of AKT mRNA and activate its degradation mechanism through regulation of several molecules, including RNA helicase of the DEAD box family, histone deacetylase, and the b-galactoside binding protein cytokine. (35)(36)(37) At the translation level, PI3K is one of the primary upstream molecules of AKT, which is a heterodimeric enzyme consisted of p110 catalytic and p85 regulatory subunits. Several reports have shown that these two subunits modify PI3K activity to further regulate its target molecule AKT, (38,39) supporting our hypothesis that solasodine probably targets p110a/b/d or p85a/b to resist PI3K/AKT signal transduction.…”

Glycobiology of cell death: when glycans and lectins govern cell fate

“…34 In contrast, galectin-1 has been proposed as a candidate inhibitor, which decreases Akt activity and induces apoptosis of breast cancer cells. 35 Recently, Mazurek and co-workers 36 documented a direct interaction between galectin-3 and TRAIL, further emphasizing the antiapoptotic properties of this lectin. When galectin-3 is expressed on colon cancer cells, it promotes the formation of a heterodimeric complex with TRAIL-R1 and TRAIL-R2 by anchoring these death receptors through glycosylation-dependent mechanisms.…”