Tumor microenvironment (TME)-driven immune suppression in B cell malignancy.

Immune checkpoint blockade antibodies and immunomodulatory drugs can unleash anti-tumor T cell immunity and mediate durable cancer regressions. However, only a fraction of patients currently respond to immunotherapy. Lymphoid malignancies are known to have clinically exploitable immune sensitivity and their intrinsic lymphoid tumor-microenvironment (TME) should make them natural targets for immunotherapy. However, accumulating evidence is showing that malignant cells engage in novel associations/interdependencies with reprogrammed immune and stromal cells in the TME that provide crucial contributions to the licencing of tumour progression and immune evasion (suppression of antitumor immune responses). In this review, we outline TME-driven contributions to the licencing of immune evasion mechanisms including the expression and activity of the immune checkpoint network, focussing on two types of B cell malignancy: indolent chronic lymphocytic leukemia (CLL) and aggressive diffuse large B-cell lymphoma (DLBCL). We also highlight recent therapeutic strategies to re-educate the TME to have anti-tumorigenic effects. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.

Imaging the spatiotemporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging, with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution, giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo.

Differential role for PAK1 and PAK4 during the invadopodia lifecycle.

PAK1 and PAK4 are members of the p-21 activated kinase family of serine/threonine kinases. PAK1 has previously been implicated in both the formation and disassembly of invasive cell protrusions, termed invadopodia. We recently reported a novel role for PAK4 during invadopodia maturation and confirmed a specific role for PAK1 in invadopodia formation; findings we will review here. Moreover, we found that PAK4 induction of maturation is delivered via interaction with the RhoA regulator PDZ-RhoGEF. We can now reveal that loss of PAK4 expression leads to changes in invadopodia dynamics. Ultimately we propose that PAK4 but not PAK1 is a key mediator of RhoA activity and provide further evidence that modulation of PAK4 expression levels leads to changes in RhoA activity.

PAK4 suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells.

Cancer cells are thought to use actin rich invadopodia to facilitate matrix degradation. Formation and maturation of invadopodia requires the co-ordained activity of Rho-GTPases, however the molecular mechanisms that underlie the invadopodia lifecycle are not fully elucidated. Previous work has suggested a formation and disassembly role for Rho family effector p-21 activated kinase 1 (PAK1) however, related family member PAK4 has not been explored. Systematic analysis of isoform specific depletion using in vitro and in vivo invasion assays revealed there are differential invadopodia-associated functions. We consolidated a role for PAK1 in the invadopodia formation phase and identified PAK4 as a novel invadopodia protein that is required for successful maturation. Furthermore, we find that PAK4 (but not PAK1) mediates invadopodia maturation likely via inhibition of PDZ-RhoGEF. Our work points to an essential role for both PAKs during melanoma invasion but provides a significant advance in our understanding of differential PAK function.

Role of p-21-activated kinases in cancer progression.

The p-21-activated kinases (PAKs) are downstream effectors of Rho GTPases Rac and Cdc42. The PAK family consists of six members which are segregated into two subgroups (Group I and Group II) based on sequence homology. Group I PAKs (PAK1-3) are the most extensively studied but there is increasing interest in the functionality of Group II PAKs (PAK4-6). The PAK family proteins are thought to play an important role in many different cellular processes, some of which have particular significance in the context of cancer progression. This review explores established and more recent data, linking the PAK family kinases to cancer progression including expression profiles, evasion of apoptosis, promotion of cell survival, and regulation of cell invasion. Finally, we discuss attempts to therapeutically target the PAK family and outline the major obstacles that still need to be overcome.

Hypoxia-induced invadopodia formation: a role for ß-PIX.

During tumour progression, oxygen tension in the microenvironment surrounding tumour cells is reduced, resulting in hypoxia. It is well established that cancer cells resist the negative effects of hypoxia by inducing angiogenesis predominantly via the activity of transcription factor hypoxia-inducible factor-1 (HIF-1). However, more recently HIF-1α has also been linked to increased invasive potential, although the molecular mechanisms remain to be defined. Invasive cancer cells are thought to employ membrane protrusions, termed invadopodia, to achieve matrix degradation. While many invadopodia components have been identified, signalling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. Indeed, the relationship between invadopodia formation and HIF-1α has not been explored. We now report that HIF-1α is a driver of invadopodia formation. Furthermore, we have identified an important, direct and novel link between the Rho family activator ß-PIX, HIF-1α and invadopodia formation. Indeed, we find that ß-PIX expression is essential for invadopodia formation. In conclusion, we identify a new HIF-1α mechanistic pathway and suggest that ß-PIX is a novel downstream signalling mediator during invadopodia formation.