Jim C. Norman

Mutant p53 Drives Invasion by Promoting Integrin Recycling

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.

Integrins: masters and slaves of endocytic transport

Since it has become clear that adhesion receptors are trafficked through the endosomal pathway and that this can influence their function, much effort has been invested in obtaining detailed descriptions of the molecular machinery responsible for internalizing and recycling integrins. New findings indicate that integrin trafficking dictates the nature of Rho GTPase signalling during cytokinesis and cell migration. Furthermore, integrins can exert control over the trafficking of other receptors in a way that drives cancer cell invasion and tumour angiogenesis.

Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors

Inhibitors of αvβ3 and αvβ5 integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic αvβ3 and αvβ5 inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering αvβ3 integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.

Integrin trafficking and the control of cell migration

In the late 1980s and early 1990s, the observation that certain integrin heterodimers are continually internalized from the plasma membrane into endosomal compartments and subsequently recycled back to the cell surface indicated that the endocytic and recycling pathways have the potential to exert minute‐to‐minute control over integrin function. This insight has prompted others to study the regulation of integrin trafficking in more detail. This review aims to summarize the findings of studies revealing the molecular mechanisms controlling integrin traffic, particularly those providing indications as to how these processes contribute to cell migration and tumour cell invasiveness.

PDGF-regulated rab4-dependent recycling of αvβ3 integrin from early endosomes is necessary for cell adhesion and spreading

Abstract Background: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. Results: The endo/exocytic cycle of αvβ3 and α5β1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, αvβ3 integrin, but not α5β1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed αvβ3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated αvβ3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for αvβ3), but adhesion to fibronectin (a ligand for α5β1 and αvβ3) was unchanged. Conclusions: We propose that growth factor-regulated, rab4-dependent recycling of αvβ3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

p53 and its mutants in tumor cell migration and invasion

In about half of all human cancers, the tumor suppressor p53 protein is either lost or mutated, frequently resulting in the expression of a transcriptionally inactive mutant p53 protein. Loss of p53 function is well known to influence cell cycle checkpoint controls and apoptosis. But it is now clear that p53 regulates other key stages of metastatic progression, such as cell migration and invasion. Moreover, recent data suggests that expression of mutant p53 is not the equivalent of p53 loss, and that mutant p53s can acquire new functions to drive cell migration, invasion, and metastasis, in part by interfering with p63 function.

Rab-coupling protein coordinates recycling of α5β1 integrin and EGFR1 to promote cell migration in 3D microenvironments

Here we show that blocking the adhesive function of αvβ3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with α5β1 integrin and drove RCP-dependent recycling of α5β1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in α5β1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of α5β1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, α5β1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of α5β1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

Mechanisms of integrin activation and trafficking

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified.

Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6.

The secreted Mycobacterium tuberculosis complex proteins CFP‐10 and ESAT‐6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP‐10 and ESAT‐6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C‐terminus of CFP‐10, which was found to be essential for binding to the surface of cells. The surface features of the CFP‐10·ESAT‐6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.

αvβ3 and α5β1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser916 is necessary for its association with αvβ3 integrin. Expression of PKD1916A or the use of mutants of β3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of αvβ3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving αvβ3 to the cell front, but by antagonizing α5β1 recycling, which, in turn, influences the cells decision to migrate with persistence or to move randomly.