Nucleoporin-93 reveals a common feature of aggressive breast cancers: robust nucleocytoplasmic transport of transcription factors.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as ß-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-ß/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport.

Serine Biosynthesis Is a Metabolic Vulnerability in IDH2-Driven Breast Cancer Progression.

Cancer-specific metabolic phenotypes and their vulnerabilities represent a viable area of cancer research. In this study, we explored the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple-negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses revealed the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration, and antioxidant defense. Genome-scale metabolic modeling identified phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction was supported by patients with IDH2-high/PHGDH-low tumors, who exhibited longer survival than patients with IDH2-high/PHGDH-high tumors. Furthermore, PHGDH inhibitors were effective in treating IDH2-high cells in vitro and in vivo. Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression. SIGNIFICANCE: These findings highlight the metabolic dependence of IDH2 on the serine biosynthesis pathway, adding an important layer to the connection between TCA cycle and glycolysis, which can be translated into novel targeted therapies.

Targeting of PYK2 Synergizes with EGFR Antagonists in Basal-like TNBC and Circumvents HER3-Associated Resistance via the NEDD4-NDRG1 Axis.

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous disease with poor prognosis and no effective targeted therapies. EGFR is highly expressed in basal-like TNBC and is considered as a potential therapeutic target. However, EGFR targeting exerts only marginal clinical benefits, possibly due to activation of compensatory signaling pathways, which are frequently associated with HER3 upregulation. Here we show that concomitant targeting of EGFR and the nonreceptor tyrosine kinases PYK2/FAK synergistically inhibits the proliferation of basal-like TNBC cells in vitro and attenuates tumor growth in a mouse xenograft model. Dual targeting of EGFR and PYK2/FAK inhibited complementary key growth and survival pathways mediated by AKT, S6K, STAT3, and ERK1/2 activation. PYK2 inhibition also abrogated HER3 upregulation in response to EGFR antagonists, thereby circumventing HER3-associated drug resistance. Mechanistically, PYK2 inhibition facilitated the proteasomal degradation of HER3 while inducing upregulation of NDRG1 (N-myc downstream regulated 1 gene). NDRG1 enhanced the interaction of HER3 with the ubiquitin ligase NEDD4, while PYK2, which interacts with NEDD4 and HER3, interfered with NEDD4-HER3 binding, suggesting that the PYK2-NDRG1-NEDD4 circuit has a critical role in receptor degradation, drug response, and resistance mechanism. Our studies offer a preclinical proof of concept for a strategy of cotargeting the EGFR and PYK2/FAK kinases to improve TNBC therapy. Cancer Res; 77(1); 86-99. ©2016 AACR.

The role of phosphatidylinositol-transfer proteins at membrane contact sites.

Phosphatidylinositol-transfer proteins (PITPs) have been initially identified as soluble factors that accelerate the monomeric exchange of either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayersin vitro They are highly conserved in eukaryotes and have been implicated in different cellular processes, including vesicular trafficking, signal transduction, and lipid metabolism. Recent studies suggest that PITPs function at membrane contact sites (MCSs) to facilitate the transport of PI from its synthesis site at the endoplasmic reticulum (ER) to various membrane compartments. In this review, we describe the underlying mechanism of PITPs targeting to MCSs, discuss their cellular roles and potential mode of action.

Cultured Mesenchymal Stem Cells Stimulate an Immune Response by Providing Immune Cells with Toll-Like Receptor 2 Ligand.

Mesenchymal stem cells (MSCs) serve as supporting and regulatory cells, by providing tissues with multiple factors and are also known for their immunosuppressive capabilities. Our laboratory had previously shown that MSCs expressed toll-like receptor (TLR) 2 and are activated by its ligand Pam3Cys. TLR2 is an important component of the innate immune system, as it recognizes bacterial lipopeptides, thus priming a pro-inflammatory immune response. This study showed that Pam3Cys attached extensively to cells of both wild-type and TLR2 deficient cultured MSCs, thus, independently of TLR2. The TLR2 independent binding occurred through the adsorption of the palmitoyl moieties of Pam3Cys. It was further showed that Pam3Cys was transferred from cultured MSCs to immune cells. Moreover, Pam3Cys provided to the immune cells induced a pro-inflammatory response in vitro and in vivo. Overall, it is demonstrated herein that a TLR2 ligand bound to MSCs also through a TLR2 independent mechanism. Furthermore, the ligand incorporated by MSCs is subsequently released to stimulate an immune response both in vitro and in vivo. It is thus suggested that during bacterial infection, stromal cells may retain a reservoir of the TLR2 ligands, in a long-term manner, and release them slowly to maintain an immune response.

PYK2 integrates growth factor and cytokine receptors signaling and potentiates breast cancer invasion via a positive feedback loop.

The involvement of ErbB family members in breast cancer progression and metastasis has been demonstrated by many studies. However, the downstream effectors that mediate their migratory and invasive responses have not been fully explored. In this study, we show that the non-receptor tyrosine kinase PYK2 is a key effector of EGFR and HER2 signaling in human breast carcinoma. We found that PYK2 is activated by both EGF and heregulin (HRG) in breast cancer cells, and positively regulates EGF/HRG-induced cell spreading, migration and invasion. PYK2 depletion markedly affects ERK1/2 and STAT3 phosphorylation in response to EGF/HRG as well as to IL8 treatment. Importantly, PYK2 depletion also reduced EGF/HRG-induced MMP9 and IL8 transcription, while IL8 inhibition abrogated EGF-induced MMP9 transcription and attenuated cell invasion. IL8, which is transcriptionally regulated by STAT3 and induces PYK2 activation, prolonged EGF-induced PYK2, STAT3 and ERK1/2 phosphorylation suggesting that IL8 acts through an autocrine loop to reinforce EGF-induced signals. Collectively our studies suggest that PYK2 is a common downstream effector of ErbB and IL8 receptors, and that PYK2 integrates their signaling pathways through a positive feedback loop to potentiate breast cancer invasion. Hence, PYK2 could be a potential therapeutic target for a subset of breast cancer patients.

PYK2 sustains endosomal-derived receptor signalling and enhances epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) is a central developmental process implicated in cancer metastasis. Here we show that the tyrosine kinase PYK2 enhances cell migration and invasion and potentiates EMT in human breast carcinoma. EMT inducer, such as EGF, induces rapid phosphorylation of PYK2 and its translocation to early endosomes where it co-localizes with EGFR and sustains its downstream signals. Furthermore, PYK2 enhances EGF-induced STAT3-phosphorylation, while phospho-STAT3 directly binds to PYK2 promoter and regulates PYK2 transcription. STAT3 and PYK2 also enhance c-Met expression, while c-Met augments their phosphorylation, suggesting a positive feedback loop between PYK2-STAT3-c-Met. We propose that PYK2 sustains endosomal-derived receptor signalling and participates in a positive feedback that links cell surface receptor(s) to transcription factor(s) activation, thereby prolonging signalling duration and potentiating EMT. Given the role of EMT in breast cancer metastasis, we also found a significant correlation between PYK2 expression, tumour grade and lymph node metastasis, thus, demonstrating the clinicopathological implication of our findings.

The lipid-transfer protein Nir2 enhances epithelial-mesenchymal transition and facilitates breast cancer metastasis.

The involvement of epithelial-mesenchymal transition (EMT) in breast cancer metastasis has been demonstrated in many studies. However, the intracellular proteins and signaling pathways that regulate EMT have not been fully identified. Here, we show that the lipid-transfer protein Nir2 (also known as PITPNM1) enhances EMT in mammary epithelial and breast cancer cells. Nir2 overexpression decreases the expression of epithelial markers and concomitantly increases the expression of mesenchymal markers, whereas silencing of Nir2 expression by small hairpin RNA (shRNA) has opposite effects. Additionally, Nir2 expression is increased during EMT and affects cell morphology, whereas Nir2 depletion attenuates growth factor-induced cell migration. These effects of Nir2 on EMT-associated processes are mainly mediated through the PI3K/AKT and the ERK1/2 pathways. Nir2 depletion also inhibits cell invasion in vitro and lung metastasis in animal models. Immunohistochemical analysis of breast cancer tissue samples reveals a correlation between high Nir2 expression and tumor grade, and Kaplan-Meier survival curves correlate Nir2 expression with poor disease outcome. These results suggest that Nir2 not only enhances EMT in vitro and breast cancer metastasis in animal models, but also contributes to breast cancer progression in human patients.

The phosphatidylinositol-transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling.

Phosphatidic acid (PA) and phosphoinositides are metabolically interconverted lipid second messengers that have central roles in many growth factor (GF)-stimulated signalling pathways. Yet, little is known about the mechanisms that coordinate their production and downstream signalling. Here we show that the phosphatidylinositol (PI)-transfer protein Nir2 translocates from the Golgi complex to the plasma membrane in response to GF stimulation. This translocation is triggered by PA formation and is mediated by its C-terminal region that binds PA in vitro. We further show that depletion of Nir2 substantially reduces the PI(4,5)P2 levels at the plasma membrane and concomitantly GF-stimulated PI(3,4,5)P3 production. Finally, we show that Nir2 positively regulates the MAPK and PI3K/AKT pathways. We propose that Nir2 through its PA-binding capability and PI-transfer activity can couple PA to phosphoinositide signalling, and possibly coordinates their local lipid metabolism and downstream signalling.

Transcription in the nucleus and mRNA decay in the cytoplasm are coupled processes.

Maintaining appropriate mRNAs levels is vital for any living cell. mRNA synthesis in the nucleus by RNA polymerase II core enzyme (Pol II) and mRNA decay by cytoplasmic machineries determine these levels. Yet, little is known about possible cross-talk between these processes. The yeast Rpb4/7 is a nucleo-cytoplasmic shuttling heterodimer that interacts with Pol II and with mRNAs and is required for mRNA decay in the cytoplasm. Here we show that interaction of Rpb4/7 with mRNAs and eventual decay of these mRNAs in the cytoplasm depends on association of Rpb4/7 with Pol II in the nucleus. We propose that, following its interaction with Pol II, Rpb4/7 functions in transcription, interacts with the transcript cotranscriptionally and travels with it to the cytoplasm to stimulate mRNA decay. Hence, by recruiting Rpb4/7, Pol II governs not only transcription but also mRNA decay.

Nucleocytoplasmic shuttling of the Rpb4p and Rpb7p subunits of Saccharomyces cerevisiae RNA polymerase II by two pathways.

Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions.