Validation of a small molecule inhibitor of PDE6D-RAS interaction with favorable anti-leukemic effects.

RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights into the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival.

Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness.

Stromal fibroblast recruitment to tumours and activation to a cancer-associated fibroblast (CAF) phenotype has been implicated in promoting primary tumour growth and progression to metastatic disease. However, the mechanisms underlying the tumour:fibroblast crosstalk that drive the intertumoural stromal heterogeneity remain poorly understood. Using in vivo models we identify Wnt7a as a key factor secreted exclusively by aggressive breast tumour cells, which induces CAF conversion. Functionally, this results in extracellular matrix remodelling to create a permissive environment for tumour cell invasion and promotion of distant metastasis. Mechanistically, Wnt7a-mediated fibroblast activation is not dependent on classical Wnt signalling. Instead, we demonstrate that Wnt7a potentiates TGFß receptor signalling both in 3D in vitro and in vivo models, thus highlighting the interaction between two of the key signalling pathways in development and disease. Importantly, in clinical breast cancer cohorts, tumour cell Wnt7a expression correlates with a desmoplastic, poor-prognosis stroma and poor patient outcome.

The 'alternative' EMT switch.

Epithelial to mesenchymal transition (EMT) is an essential process in embryonic development and is aberrantly induced in many disease settings. Work carried out by Chonghui Cheng's laboratory addressed the involvement of alternative RNA splicing in EMT and its link to tumour progression. They describe a switch in CD44 expression from variant isoform(s) to the standard isoform and showed, for the first time, that this is required for normal epithelial cells to undergo EMT. In addition, they link expression of the CD44 standard isoform with high-grade breast cancer and to activation of the phosphoinositide 3-kinase/Akt pathway and apoptosis resistance in a mouse model of recurrent disease.

CD44 is overexpressed in basal-like breast cancers but is not a driver of 11p13 amplification.

Overexpression and alternative splicing of CD44 have been implicated in tumour progression. Here we describe the identification of a high level amplification of human 11p13, encompassing the CD44 gene, in primary breast cancers and cell lines and test whether CD44 acts as the driver of this amplicon. aCGH analysis revealed 11p13 amplification in 3% (3/100) of primary breast carcinomas and in two cell lines. The minimal region of amplification was 34.38-37.62 Mb. Amplification was confirmed by dual-colour FISH in these cell lines and further validated by CISH in an independent tumour cohort. CD44 expression in primary breast cancers was significantly associated with features of basal-like breast cancer. Detection of CD44 expression in breast cancer cell lines confirmed moderate to high expression in basal-like cell lines and minimal expression in luminal cell lines. In both, primary breast cancers and cell lines, 11p13 amplification was associated with high levels of CD44 mRNA expression. CD44 alternative splicing was detected in four of nine cell lines and in tumour samples, irrespective of the amplification status. RNAi mediated knock down of CD44 failed to reveal an increased dependence on CD44 expression for proliferation or survival in amplified cell lines. Given that expression of CD44 is not an absolute requirement for the survival of cells harbouring CD44 gene amplification, CD44 is unlikely to be a driver of the 11p13 amplicon.

CD44v6 dependence of premetastatic niche preparation by exosomes.

The metastasizing capacity of the rat pancreatic adenocarcinoma BSp73ASML (ASML(wt)) is strikingly reduced by a knockdown of CD44v4-v7 (ASML(kd)). We used this model to analyze the role of the CD44 variant isoform (CD44v) in (pre)metastatic niche formation. Intrafootpad injections of ASML(wt)-, but not ASML(kd)-conditioned medium (CM), strongly promote settlement of ASML(kd) cells in lymph nodes and lung. Fractionation of CM revealed a contribution by a soluble matrix and exosomes, where the CD44v6-containing ASML(wt)-soluble fraction can complement ASML(kd)-exosomes, but not vice versa. This implies that exosomes are the final actors, are CD44v-independent, but require a soluble matrix, which depends on CD44v. Analyzing the composition revealed that only the ASML(wt)-matrix contains c-Met and urokinase-type plasminogen activator receptor. In vitro, mostly ASML(wt)-exosomes promote proliferation and induce gene expression in metastatic organ cells. However, in vivo corresponding changes in the (pre) metastatic organ are only observed when both, exosomes plus the soluble matrix, are provided. Thus, neither CD44v nor exosomes alone suffice for (pre)metastatic niche formation. Instead, CD44v suffices for assembling a soluble matrix, which allows exosomes, independent of their origin from poorly or highly metastatic cells, to modulate (pre) metastatic organ cells for tumor cell embedding and growth.

CD44 variant isoforms promote metastasis formation by a tumor cell-matrix cross-talk that supports adhesion and apoptosis resistance.

CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44v(kd)) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASML(wt)) were established. ASML-CD44v(kd) clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44v(kd) cells (ASML-CD44v(rsc)) restores the metastatic potential. The following major differences in ASML(wt), ASML-CD44v(kd), and ASML-CD44v(rsc) clones were observed: (a) ASML(wt) cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44v(kd) cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASML(wt) cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44v(kd) cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44v(rsc) cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation.

CD44 and EpCAM: cancer-initiating cell markers.

Embryonic stem cells are immortal, can self renew, and differentiate into all cells of the body. The adult organism maintains adult stem cells in regenerative organs that can differentiate into all cells of the respective organ. Virchow's hypothesis that cancer may arise from embryonic-like cells has received strong support, as it was demonstrated that tumors contain few cells, known as cancer stem or cancer-initiating cells (CIC), that account for primary and metastatic tumor growth. CIC are mostly defined by expression of CIC-markers that are associated and correlated with the potential of CIC to grow in xenogeneic mice. CIC marker profiles have been elaborated for many tumors, with several markers as CD24, CD44, CD133, CD166, EpCAM, and some integrins, being expressed by tumors of different histological type. Their function in promoting CIC maintenance and activity is largely unknown. The fate of stem cells, determined by their position, is minutely regulated by few adjacent cells creating a niche. CIC also require a niche, mostly for settlement and growth in distant organs. This so called pre-metastatic niche is initiated by the primary tumor before metastasizing cell arrival. How do CIC prepare the pre-metastatic niche? Cancer cells secrete a matrix that serves a cross-talk with surrounding tissues. Additionally, cancer cells can abundantly deliver exosomes, which function as long-distance intercellular communicators. Studies on a rat pancreatic adenocarcinoma support our hypothesis that tumor-derived matrix and exosomes are the main actors in forming the pre-metastatic niche with CIC markers being engaged in matrix preparation and/or exosome delivery.

A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression.

High expression of EpCAM and the tetraspanin CO-029 has been associated with colorectal cancer progression. However, opposing results have been reported on CD44 variant isoform v6 (CD44v6) expression. We recently noted in rat gastrointestinal tumors that EpCAM, claudin-7, CO-029, and CD44v6 were frequently coexpressed and could form a complex. This finding suggested the possibly that the complex, rather than the individual molecules, could support tumor progression. The expression of EpCAM, claudin-7, CO-029, and CD44v6 expression was evaluated in colorectal cancer (n = 104), liver metastasis (n = 66), and tumor-free colon and liver tissue. Coexpression and complex formation of the molecules was correlated with clinical variables and apoptosis resistance. EpCAM, claudin-7, CO-029, and CD44v6 expression was up-regulated in colon cancer and liver metastasis. Expression of the four molecules did not correlate with tumor staging and grading. However, coexpression inversely correlated with disease-free survival. Coexpression was accompanied by complex formation and recruitment into tetraspanin-enriched membrane microdomains (TEM). Claudin-7 contributes to complex formation inasmuch as in the absence of claudin-7, EpCAM hardly associates with CO-029 and CD44v6 and is not recruited into TEMs. Notably, colorectal cancer lines that expressed the EpCAM/claudin-7/CO-029/CD44v6 complex displayed a higher degree of apoptosis resistance than lines devoid of any one of the four molecules. Expression of EpCAM, claudin-7, CO-029, and CD44v6 by themselves cannot be considered as prognostic markers in colorectal cancer. However, claudin-7-associated EpCAM is recruited into TEM and forms a complex with CO-029 and CD44v6 that facilitates metastasis formation.

CD44 variant isoforms associate with tetraspanins and EpCAM.

The metastasizing subline of the rat pancreatic adenocarcinoma BSp73 expresses a set of membrane molecules, the combination of which has not been detected on non-metastasizing tumor lines. Hence, it became of interest whether these molecules function independently or may associate and exert specialized functions as membrane complexes. Separation of CD44v4-v7 containing membrane complexes in mild detergent revealed an association with the alpha3 integrin, annexin I, EpCAM, and the tetraspanins D6.1A and CD9. EpCAM and the tetraspanins associate selectively with CD44 variant (CD44v), but not with the CD44 standard (CD44s) isoform. The complexes are found in glycolipid-enriched membrane (GEM) microdomains, which are dissolved by stringent detergents, but the complexes are not destroyed by methyl-beta-cyclodextrin (MbetaCD) treatment, which implies that complex formation does not depend on a lipid-rich microenvironment. However, a complex-associated impact on cell-matrix and cell-cell adhesion as well as on resistance towards apoptosis essentially depended on the location in GEMs. Thus, CD44v-specific functions may well be brought about by complex formation of CD44v with EpCAM, the tetraspanins, and the alpha3 integrin. Because CD44v4-v7-EpCAM complex-specific functions strictly depended on the GEM localization, linker or signal-transducing molecules associating with the complex are likely located in GEMs.

Xenopus marginal coil (Xmc), a novel FGF inducible cytosolic coiled-coil protein regulating gastrulation movements.

Gastrulation in vertebrates is a highly dynamic process driven by convergent extension movements of internal mesodermal cells, under the regulatory activity of the Spemann-Mangold or gastrula organizer. In a large-scale screen for genes expressed in the organizer, we have isolated a novel gene, termed Xmc, an acronym for Xenopus marginal coil. Xmc encodes a protein containing two widely spaced evolutionarily non-conserved coiled coils. Xmc protein is found in vesicular aggregates in the cytoplasm and associated with the inner plasma membrane. We show that Xmc is expressed in a dynamic fashion around the blastoporal circumference, in mesodermal cells undergoing morphogenetic movements, in a pattern similar to FGF target genes. Likewise, Xmc expression can be induced by ectopic XeFGF signaling and the early mesodermal expression is dependent on FGF receptor-mediated signaling. Morpholino-mediated translational 'knock-down' of Xmc results in embryos that display a reduced elongation of the antero-posterior axis and in a pronounced inhibition of morphogenetic movements in embryos and dorsal marginal zone explants. Xmc loss-of-function does not interfere with mesoderm induction or maintenance per se. Our results suggest that Xmc is a novel FGF target gene that is required for morphogenetic movements during gastrulation in Xenopus.