Topi A. Tervonen

Tumor suppressor function of Liver kinase B1 (Lkb1) is linked to regulation of epithelial integrity

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy with c-Myc imply that Lkb1 loss facilitates oncogenic proliferation by releasing epithelial cells from structural BM boundaries.

Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis

Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Here, we show that Myc-induced altered glutamine metabolism involves ATP depletion and activation of the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. Under influence of Myc, AMPK-stabilized tumor suppressor protein p53 accumulates in the mitochondria and interacts with the protein complex comprised of B-cell lymphoma 2 (Bcl-2) antagonist/killer (BAK) and Bcl2-like 1 (Bcl-xL). Mitochondrial p53 induces conformational activation of proapoptotic Bak without disrupting the Bak–Bcl-xL interaction. Further liberation of Bak specifically from the p53-activated Bak–Bcl-xL complex leads to spontaneous oligomerization of Bak and apoptosis. Thus, Myc-induced metabolic changes are coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak, demonstrating a cell-intrinsic mechanism to counteract uncontrolled proliferation.

Aberrant differentiation of glutamatergic cells in neocortex of mouse model for fragile X syndrome

The lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. Our previous studies revealed alterations in the differentiation of FMRP-deficient neural progenitors. Here, we show abnormalities in neurogenesis in the mouse and human embryonic FMRP-deficient brain as well as after in utero transfection of I304N mutated FMRP, which acts in a dominant negative manner in the wild-type mouse brain. Progenitors accumulated abnormally in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) mouse neocortex. An increased density of cells expressing sequentially an intermediate progenitor marker, T-box transcription factor (Tbr2), and a postmitotic neuron marker, T-brain 1 (Tbr1), indicated that the differentiation to glutamatergic cell lineages was particularly disturbed. These abnormalities were associated with an increased density of pyramidal cells of the layer V in the early postnatal neocortex suggesting a role for FMRP in the regulation of the differentiation of neocortical glutamatergic neurons.

Overexpression of a truncated TrkB isoform increases the proliferation of neural progenitors

The truncated isoform of TrkB, TrkB.T1, has been shown to be expressed in the neurogenic region of rodent brain. TrkB.T1 lacks tyrosine kinase activity and it may modify the action of the full‐length TrkB. We show here that the full‐length TrkB and TrkB.T1 are expressed at the same relative expression levels in mouse neural progenitor cell cultures. The number of neurosphere‐forming progenitors was reduced and apoptosis increased in neurospheres generated from mice overexpressing TrkB.T1 when compared with wild‐type neurospheres. The proliferation of the transgenic neural progenitors was increased, as indicated by the larger average diameter of spheres (140% of control), the increased cell growth in an MTT assay (137% of control) and the faster rate of 3H‐thymidine incorporation (128% of control) in the transgenic cell cultures than in controls. The proliferation of neural progenitors was also increased after lentivirus‐mediated TrkB.T1 overexpression. A significant increase in 3H‐thymidine incorporation (119% of control) and the average diameter of spheres (112% of control) in the TrkB.T1‐transduced neurospheres compared with neurospheres transduced with the control vectors confirmed the role of TrkB.T1 in proliferation of neural progenitor. When induced to differentiate, progenitors overexpressing TrkB.T1 generated two to three times more neurons than did wild‐type cells. The increase in the number of neurons correlated with an increase in the number of apoptotic cells (two‐fold) at these time points. The data indicate that changes in the relative expression levels of different TrkB isoforms influence the replicative capacity of neural progenitors.

Faulty epithelial polarity genes and cancer.

Epithelial architecture is formed in tissues and organs when groups of epithelial cells are organized into polarized structures. The epithelial function and integrity as well as signaling across the epithelial layer is orchestrated by apical junctional complexes (AJCs), which are landmarks for PAR/CRUMBS and lateral SCRIB polarity modules and by dynamic interactions of the cells with underlying basement membrane (BM). These highly organized epithelial architectures are demolished in cancer. In all advanced epithelial cancers, malignant cells have lost polarity and connections to the basement membrane and they have become proliferative, motile, and invasive. Clearly, loss of epithelial integrity associates with tumor progression but does it contribute to tumor development? Evidence from studies in Drosophila and recently also in vertebrate models have suggested that even the oncogene-driven enforced cell proliferation can be conditional, dependant on the influence of cell-cell or cell-microenvironment contacts. Therefore, loss of epithelial integrity may not only be an obligate consequence of unscheduled proliferation of malignant cells but instead, malignant epithelial cells may need to acquire capacity to break free from the constraints of integrity to freely and autonomously proliferate. We discuss how epithelial polarity complexes form and regulate epithelial integrity, highlighting the roles of enzymes Rho GTPases, aPKCs, PI3K, and type II transmembrane serine proteases (TTSPs). We also discuss relevance of these pathways to cancer in light of genetic alterations found in human cancers and review molecular pathways and potential pharmacological strategies to revert or selectively eradicate disorganized tumor epithelium.

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

The PAR clan of polarity regulating genes was initially discovered in a genetic screen searching for genes involved in asymmetric cell divisions in the Caenorhabditis elegans embryo. Today, investigations in worms, flies and mammals have established PAR proteins as conserved and fundamental regulators of animal cell polarization in a broad range of biological phenomena requiring cellular asymmetries. The human homologue of invertebrate PAR-4, a serine–threonine kinase LKB1/STK11, has caught attention as a gene behind Peutz–Jeghers polyposis syndrome and as a bona fide tumour suppressor gene commonly mutated in sporadic cancer. LKB1 functions as a master regulator of AMP-activated protein kinase (AMPK) and 12 other kinases referred to as the AMPK-related kinases, including four human homologues of PAR-1. The role of LKB1 as part of the energy sensing LKB1-AMPK module has been intensively studied, whereas the polarity function of LKB1, in the context of homoeostasis or cancer, has gained less attention. Here, we focus on the PAR-4 identity of LKB1, discussing the weight of evidence indicating a role for LKB1 in regulation of cell polarity and epithelial integrity across species and highlight recent investigations providing new insight into the old question: does the PAR-4 identity of LKB1 matter in cancer?

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.

Par6G suppresses cell proliferation and is targeted by loss-of-function mutations in multiple cancers

Differentiated epithelial structure communicates with individual constituent epithelial cells to suppress their proliferation activity. However, the pathways linking epithelial structure to cessation of the cell proliferation machinery or to unscheduled proliferation in the context of tumorigenesis are not well defined. Here we demonstrate the strong impact of compromised epithelial integrity on normal and oncogenic Myc-driven proliferation in three-dimensional mammary epithelial organoid culture. Systematic silencing of 34 human homologs of Drosophila genes, with previously established functions in control of epithelial integrity, demonstrates a role for human genes of apico-basal polarity, Wnt and Hippo pathways and actin dynamics in regulation of the size, integrity and cell proliferation in organoids. Perturbation of these pathways leads to diverse functional interactions with Myc: manifested as a RhoA-dependent synthetic lethality and Par6-dependent effects on the cell cycle. Furthermore, we show a role for Par6G as a negative regulator of the phosphatidylinositol 3′-kinase/phosphoinositide-dependent protein kinase 1/Akt pathway and epithelial cell proliferation and evidence for frequent inactivation of Par6G gene in epithelial cancers. The findings demonstrate that determinants of epithelial structure regulate the cell proliferation activity via conserved and cancer-relevant regulatory circuitries, which are important for epithelial cell cycle restriction and may provide new targets for therapeutic intervention.

Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform

Hepsin is a membrane-anchored serine protease whose role in hepatocyte growth factor (HGF) signaling and epithelial integrity makes it a target of therapeutic interest in carcinogenesis and metastasis. Using an integrated design, synthesis, and screening platform, we were able to rapidly develop potent and selective inhibitors of hepsin. In progressing from the initial hit 7 to compound 53, the IC50 value against hepsin was improved from ∼1 μM to 22 nM, and the selectivity over urokinase-type plasminogen activator (uPA) was increased from 30-fold to >6000-fold. Subsequent in vitro ADMET profiling and cellular studies confirmed that the leading compounds are useful tools for interrogating the role of hepsin in breast tumorigenesis.

Analyzing the Type II Transmembrane Serine Protease Hepsin-Dependent Basement Membrane Remodeling in 3D Cell Culture

Breakdown of the basement membrane is a key step that precedes tumor invasion, and accumulating evidence suggests a key role for the type II transmembrane proteases (TTSPs) in that process. Overexpression of a TTSP hepsin characterizes many solid cancers, including prostate, breast, and ovarian cancer, and in experimental tumor models, the elevated proteolytic activity of hepsin simultaneously activates several growth factors and cleaves basement membrane protein laminin-332, which is an essential component of the cell-basement membrane junction hemidesmosome. These hepsin-dependent molecular events associate with dramatic loss of basement membrane integrity in mouse tumor models and in three-dimensional (3D) epithelial culture. In particular, the 3D culture systems offer unprecedented possibilities to clarify the mechanistic basis of destructive interactions between out-of-control serine protease activity and the basement membrane structure. Here, we describe how to establish 3D mammary epithelial culture in an exogenous basement membrane-free egg white matrix and provide a protocol for quantitative analysis of the impact of hepsin on laminin-332 and its hemidesmosomal receptor α6-integrin by means of confocal microscopy imaging. These protocols were established to facilitate studies aiming to decipher the exact role of oncogenic proteases in tumor invasion processes and to identify novel therapeutic agents able to intervene these cancer critical processes.