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Mutant KRAS, as well as other mutant driver genes and epidriver genes, is a dominant determinant of resistance to cancer therapeutics. The recent introduction of targeting therapies based on drugs that inhibit the kinase catalytic function of nodal points along the Ras/extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol-3-kinase (PI3K)/Akt cascades is meeting with limited success. Against this background, recent evidence shows that the β-galactoside-binding protein (βGBP) molecule, a physiological PI3K inhibitor, is a potent inducer of apoptosis in KRAS-mutant cancer cells (along with other aggressive cancer cells of different genetic makeup) and that it is therapeutically effective in vivo. Absence of p53 or phosphatase and tensin homolog (PTEN) tumor suppressor function or added activating PI3K mutations does not affect βGBP function. In contrast to the concept of one drug against one target, βGBP operates through alternative physiological routes., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Activating mutations in Kras are the most frequent mutations in human cancer. They define a subset of patients who do not respond to current therapies and for whom prognosis is poor. Oncogenic Kras has been shown to deregulate numerous signaling pathways of which the most intensively studied are the Ras/extracellular signal-regulated kinase cascade and the phosphoinositide 3-kinase (PI3K)/Akt cascade. However, to date, there are no effective targeted therapies in the clinic against Kras-mutant cancers. Here, we report that the β-galactoside-binding protein (βGBP) cytokine, a physiologic inhibitor of class I PI3Ks, is a potent activator of apoptosis in Kras-mutant colorectal cancer cells, even when coharboring mutant-activated PIK3CA. Our study unveils an elective route to intrinsic and extrinsic apoptosis, which involves the cytoskeleton. Early events are inhibition of PI3K activity and Rac-independent actin rearrangement assignable to phosphoinositide changes at the plasma membrane. Cyclin E deregulation, arrest of DNA synthesis, and checkpoint kinase 2 activation underscore events critical to the activation of an intrinsic apoptotic program. Clustering of CD95/Fas death receptors underscore events critical to the activation of extrinsic apoptosis. In nude mice, we present the first evidence that xenograft tumor development is strongly inhibited by Hu-r-βGBP. Taken together, our results open a new therapeutic opportunity to a subset of patients refractive to current treatments. This first demonstration of therapeutic efficacy against Kras-mutant colon cancer suggests that Hu-r-βGBP may also be therapeutically effective against other cancers harboring activating Ras mutations as well as PIK3CA mutations., (©2012 AACR.)

Introduction: Phosphoinositide 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 beta galactoside binding protein (betaGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, betaGBP 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., Methods: Aggressive 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 betaGBP 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., Results: In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor, functional inhibition of the catalytic activity of PI3K by the betaGBP 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 betaGBP 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., Conclusions: betaGBP, 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, betaGBP has the potential to be safely tested in clinical trials.

This review highlights the discovery of beta-galactoside binding protein (betaGBP), an antiproliferative cytokine, as a potent and selective anticancer agent. Unlike drugs designed to block receptor tyrosine kinases or specific control points along signaling pathways, betaGBP does not harm normal cells. As a physiological effector molecule, betaGBP can selectively induce death in cancer cells by enforcing its regulatory functions to which normal and cancer cells respond differently, and thus exploit the genetic and molecular deviations developed by cancer cells.

We report here that beta-galactoside binding protein (betaGBP), an antiproliferative cytokine which can program cancer cells to undergo apoptosis, exhibits equal therapeutic efficacy against cancer cells that display diverse mechanisms of drug resistance and against their parental cells. The mechanisms of drug resistance in the cancer cells that we have examined include overexpression of P-glycoprotein, increased efficiency of DNA repair, and altered expression and mutation in the topoisomerase I and II enzymes. We also report that betaGBP exerted its effect by arresting the cells in S phase prior to the activation of programmed cell death. The uniquely similar profile of response to betaGBP by these drug-resistant cells and their parental cells extends the therapeutic potential of this cytokine in the treatment of cancers and offers a promising alternative to patients whose tumors are refractory to the currently available cadre of chemotherapeutic agents.

Cancer therapies based on drugs designed to interfere with specific targets within the molecular circuitry of cancer cells are currently under intense experimentation. Our strategy is based on the use of a naturally occurring immunomolecule which can selectively kill cancer cells, based on its ability to exploit genetic differences between normal and cancer cells. The betaGBP cytokine has previously been shown to negatively regulate the cell cycle by blocking cells in late S phase. In tumour cells, but not in normal cells, the S phase block has been shown to be followed by apoptosis. Mechanisms involved in S phase arrest have been pinpointed to downregulation of signalling and altered expression of cell cycle controller proteins, including E2F1, a transcription factor with ability to play a part in apoptosis. Here we discuss the use of betaGBP within the context of cancer surveillance and cancer therapeutics focussing on E2F1 as one mechanistic aspect relevant to betaGBP's selective induction of programmed cell death in cancer.