Inhibition of lung tumorigenesis by metformin is associated with decreased plasma IGF-I and diminished receptor tyrosine kinase signaling.

Metformin is the most commonly prescribed drug for type II diabetes and is associated with decreased cancer risk. Previously, we showed that metformin prevented tobacco carcinogen (NNK)-induced lung tumorigenesis in a non-diabetic mouse model, which was associated with decreased IGF-I/insulin receptor signaling but not activation of AMPK in lung tissues, as well as decreased circulating levels of IGF-I and insulin. Here, we used liver IGF-I-deficient (LID) mice to determine the importance of IGF-I in NNK-induced lung tumorigenesis and chemoprevention by metformin. LID mice had decreased lung tumor multiplicity and burden compared with wild-type (WT) mice. Metformin further decreased lung tumorigenesis in LID mice without affecting IGF-I levels, suggesting that metformin can act through IGF-I-independent mechanisms. In lung tissues, metformin decreased phosphorylation of multiple receptor tyrosine kinases (RTK) as well as levels of GTP-bound Ras independently of AMPK. Metformin also diminished plasma levels of several cognate ligands for these RTKs. Tissue distribution studies using [(14)C]-metformin showed that uptake of metformin was high in liver but four-fold lower in lungs, suggesting that the suppression of RTK activation by metformin occurs predominantly via systemic, indirect effects. Systemic inhibition of circulating growth factors and local RTK signaling are new AMPK-independent mechanisms of action of metformin that could underlie its ability to prevent tobacco carcinogen-induced lung tumorigenesis.

Repositioning metformin for cancer prevention and treatment.

Metformin is the most commonly prescribed drug for type 2 diabetes (T2DM). Retrospective studies show that metformin is associated with decreased cancer risk. This historical correlation has driven vigorous research campaigns to determine the anticancer mechanisms of metformin. Consolidating the preclinical data is a challenge because unanswered questions remain concerning relevant mechanisms, bioavailability, and genetic factors that confer metformin sensitivity. Perhaps the most important unanswered question is whether metformin has activity against cancer in non-diabetics. In this review we highlight the proposed mechanisms of metformin action in cancer and discuss ongoing clinical trials with metformin in cancer. Improved understanding of these issues will increase the chances for successful application of metformin as an inexpensive, well-tolerated, and effective anticancer agent.

Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity.

As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55(-/-)) mouse model, and demonstrate that p55(-/-) neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55(-/-) neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55(-/-) neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase gamma (PI3Kgamma), responsible for chemoattractant-induced synthesis of PIP(3) and Akt phosphorylation, is unperturbed in p55(-/-) neutrophils. Although the total amount of PIP(3) is normal in p55(-/-) neutrophils, PIP(3) is diffusely localized and forms punctate aggregates in activated p55(-/-) neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kgamma activity.

Identification of erythrocyte p55/MPP1 as a binding partner of NF2 tumor suppressor protein/Merlin.

Neurofibromatosis type 2 is an inherited disorder characterized by the development of benign and malignant tumors on the auditory nerves and central nervous system with symptoms including hearing loss, poor balance, skin lesions, and cataracts. Here, we report a novel protein-protein interaction between NF2 protein (merlin or schwannomin) and erythrocyte p55, also designated as MPP1. The p55 is a conserved scaffolding protein with postulated functions in cell shape, hair cell development, and neural patterning of the retina. The FERM domain of NF2 protein binds directly to p55, and surface plasmon resonance analysis indicates a specific interaction with a kD value of 3.7 nM. We developed a specific monoclonal antibody against human erythrocyte p55, and found that both p55 and NF2 proteins are colocalized in the non-myelin-forming Schwann cells. This finding suggests that the p55-NF2 protein interaction may play a functional role in the regulation of apico-basal polarity and tumor suppression pathways in non-erythroid cells.

Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells.

Direct physical linkage of MAGUKs to the actin cytoskeleton was first established by the interaction of erythrocyte p55 with the FERM domain of protein 4.1R. Subsequently, it was reported that p55 binds to a 51-amino acid peptide, encoded by exon 10, located within the FERM domain of protein 4.1R. In this study, we investigated the nature of the p55-FERM domain binding interface and show that p55 binds to a second 35-amino acid peptide, encoded by an alternatively spliced exon 5, located within the FERM domain of protein 4.1R. Competition and Surface Plasmon Resonance-binding measurements suggest that the peptides encoded by exons 5 and 10 bind to independent sites within the D5 domain of p55. Interestingly, the full length 135 kDa isoform of protein 4.1R containing both exons 5 and 10 was targeted exclusively to the plasma membrane of epithelial cells whereas the same isoform without exon 5 completely lost its membrane localization capacity. Together, these results indicate that p55 binds to two distinct sites within the FERM domain, and the alternatively spliced exon 5 is necessary for the membrane targeting of protein 4.1R in epithelial cells. Since sequences similar to the exon 5-peptide of protein 4.1R and D5 domain of p55 are conserved in many proteins, our findings suggest that a similar mechanism may govern the membrane targeting of other FERM domain containing proteins.