Protein Kinase C–Dependent Mobilization of the α6β4 Integrin from Hemidesmosomes and Its Association with Actin-Rich Cell Protrusions Drive the Chemotactic Migration of Carcinoma Cells

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the α6β4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express α6β4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the α6β4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis (∼1 ng/ml), the α6β4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained α6β4 in association with F-actin. Importantly, we demonstrate that this mobilization of α6β4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-α and that it is associated with the phosphorylation of the β4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin–rich cell protrusions that mediate α6β4-dependent cell movement but also the disruption of α6β4-containing hemidesmosomes by protein kinase C

NFAT promotes carcinoma invasive migration through glypican-6

Invasive migration of carcinoma cells is a prerequisite for the metastatic dissemination of solid tumours. Numerous mechanisms control the ability of cancer cells to acquire a motile and invasive phenotype, and subsequently degrade and invade the basement membrane. Several genes that are up-regulated in breast carcinoma are responsible for mediating the metastatic cascade. Recent studies have revealed that the NFAT (nuclear factor of activated T-cells) is a transcription factor that is highly expressed in aggressive breast cancer cells and tissues, and mediates invasion through transcriptional induction of pro-invasion and migration genes. In the present paper we demonstrate that NFAT promotes breast carcinoma invasion through induction of GPC (glypican) 6, a cell-surface glycoprotein. NFAT transcriptionally regulates GPC6 induction in breast cancer cells and binds to three regulatory elements in the GPC6 proximal promoter. Expression of GPC6 in response to NFAT signalling promotes invasive migration, whereas GPC6 silencing with shRNA (small-hairpin RNA) potently blocks this phenotype. The mechanism by which GPC6 promotes invasive migration involves inhibition of canonical β-catenin and Wnt signalling, and up-regulation of non-canonical Wnt5A signalling leading to the activation of JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Thus GPC6 is a novel NFAT target gene in breast cancer cells that promotes invasive migration through Wnt5A signalling

Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of ~63 and 69 kDa (y- and a-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole- 2,5-dione), the intracellular Ca^(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5- chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4- methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N- (6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca^(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases

Identifying and Targeting Sporadic Oncogenic Genetic Aberrations in Mouse Models of Triple-Negative Breast Cancer

Triple-negative breast cancers (TNBC) are genetically characterized by aberrations in TP53 and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify somatic genetic alterations in mouse models of TNBCs driven by loss of Trp53 alone or in combination with Brca1. Amplifications or translocations that resulted in elevated oncoprotein expression or oncoprotein-containing fusions, respectively, as well as frameshift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. Although the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNA-seq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine–guided TNBC treatment.National Institutes of Health (U.S.) (Grant R35 CA197588)National Institutes of Health (U.S.) (Grant R01 GM041890)National Institutes of Health (U.S.) (Grant PSOC U54 CA210184)Breast Cancer Research Foundation (award BCRF-16-021)Jon and Mindy Gray FoundationEntertainment Industry Foundation. Stand Up to Cancer Colorectal Cancer Dream Team (Tranlational Research Grant No. SU2C-AACR-DT22-17)Susan Komen postdoctoral fellowshipBreast Cancer AllianceNovo Nordisk STAR Postdoctoral Fellowshi

Pentraxin-3 is a PI3K signaling target that promotes stem cell–like traits in basal-like breast cancers

Basal-like breast cancers (BLBCs) exhibit hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway because of the frequent mutational activation of the PIK3CA catalytic subunit and the genetic loss of its negative regulators PTEN (phosphatase and tensin homolog) and INPP4B (inositol polyphosphate-4-phosphatase type II). However, PI3K inhibitors have had limited clinical efficacy in BLBC management because of compensatory amplification of PI3K downstream signaling loops. Therefore, identification of critical PI3K mediators is paramount to the development of effective BLBC therapeutics. Using transcriptomic analysis of activated PIK3CA-expressing BLBC cells, we identified the gene encoding the humoral pattern recognition molecule pentraxin-3 (PTX3) as a critical target of oncogenic PI3K signaling. We found that PTX3 abundance is stimulated, in part, through AKT- and nuclear factor κB (NF-κB)-dependent pathways and that presence of PTX3 is necessary for PI3K-induced stem cell-like traits. We further showed that PTX3 expression is greater in tumor samples from patients with BLBC and that it is prognostic of poor patient survival. Our results thus reveal PTX3 as a newly identified PI3K-regulated biomarker and a potential therapeutic target in BLBC

pVHL suppresses kinase activity of Akt in a proline-hydroxylation-dependent manner

Activation of the serine-threonine kinase Akt promotes the survival and proliferation of various cancers. Hypoxia promotes the resistance of tumor cells to specific therapies. We therefore explored a possible link between hypoxia and Akt activity. We found that Akt was prolyl-hydroxylated by the oxygen-dependent hydroxylase EglN1. The von Hippel–Lindau protein (pVHL) bound directly to hydroxylated Akt and inhibited Akt activity. In cells lacking oxygen or functional pVHL, Akt was activated to promote cell survival and tumorigenesis. We also identified cancer-associated Akt mutations that impair Akt hydroxylation and subsequent recognition by pVHL, thus leading to Akt hyperactivation. Our results show that microenvironmental changes, such as hypoxia, can affect tumor behaviors by altering Akt activation, which has a critical role in tumor growth and therapeutic resistance

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling

LINC00520 is induced by Src, STAT3, and PI3K and plays a functional role in breast cancer

Long non-coding RNAs (lncRNAs) have been implicated in normal cellular homeostasis as well as pathophysiological conditions, including cancer. Here we performed global gene expression profiling of mammary epithelial cells transformed by oncogenic v-Src, and identified a large subset of uncharacterized lncRNAs potentially involved in breast cancer development. Specifically, our analysis revealed a novel lncRNA, LINC00520 that is upregulated upon ectopic expression of oncogenic v-Src, in a manner that is dependent on the transcription factor STAT3. Similarly, LINC00520 is also increased in mammary epithelial cells transformed by oncogenic PI3K and its expression is decreased upon knockdown of mutant PIK3CA. Additional expression profiling highlight that LINC00520 is elevated in a subset of human breast carcinomas, with preferential enrichment in the basal-like molecular subtype. ShRNA-mediated depletion of LINC00520 results in decreased cell migration and loss of invasive structures in 3D. RNA sequencing analysis uncovers several genes that are differentially expressed upon ectopic expression of LINC00520, a significant subset of which are also induced in v-Src-transformed MCF10A cells. Together, these findings characterize LINC00520 as a lncRNA that is regulated by oncogenic Src, PIK3CA and STAT3, and which may contribute to the molecular etiology of breast cancer