Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.

Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances.

Proteogenomic Approaches for the Identification of NF1/Neurofibromin-depleted Estrogen Receptor-positive Breast Cancers for Targeted Treatment.

Unlabelled: NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors.

Elevated NRAS expression during DCIS is a potential driver for progression to basal-like properties and local invasiveness.

Background: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive.

Neurofibromin Is an Estrogen Receptor-α Transcriptional Co-repressor in Breast Cancer.

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER breast cancer.

Induction of N-Ras degradation by flunarizine-mediated autophagy.

Ras GTPases are powerful drivers for tumorigenesis, but directly targeting Ras for treating cancer remains challenging. The growth and transforming activity of the aggressive basal-like breast cancer (BLBC) are driven by N-Ras. To target N-Ras in BLBC, this study screened existing pharmacologically active compounds for the new ability to induce N-Ras degradation, which led to the identification of flunarizine (FLN), previously approved for treating migraine and epilepsy.

Author Correction: The prognostic effects of somatic mutations in ER-positive breast cancer.

The original version of this Article contained errors in the depiction of confidence intervals in the NF1 BCSS data illustrated in Figure 3b. These have now been corrected in both the PDF and HTML versions of the Article. The incorrect version of Figure 3b is presented in the associated Author Correction.

The prognostic effects of somatic mutations in ER-positive breast cancer.

Here we report targeted sequencing of 83 genes using DNA from primary breast cancer samples from 625 postmenopausal (UBC-TAM series) and 328 premenopausal (MA12 trial) hormone receptor-positive (HR+) patients to determine interactions between somatic mutation and prognosis. Independent validation of prognostic interactions was achieved using data from the METABRIC study. Previously established associations between MAP3K1 and PIK3CA mutations with luminal A status/favorable prognosis and TP53 mutations with Luminal B/non-luminal tumors/poor prognosis were observed, validating the methodological approach.

Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation.

Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells.

Int6 reduction activates stromal fibroblasts to enhance transforming activity in breast epithelial cells.

Background: The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6's role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts.

Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers.

Characterizing the genetic alterations leading to the more aggressive forms of oestrogen receptor-positive (ER+) breast cancers is of critical significance in breast cancer management. Here we identify recurrent rearrangements between the oestrogen receptor gene ESR1 and its neighbour CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal B tumours, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170-positive tumours.

A bimolecular fluorescent complementation screen reveals complex roles of endosomes in Ras-mediated signaling.

While Ras GTPases are best known for mediating growth factor signaling on the plasma membrane, these proteins also have surprisingly complex activities in the endosome. Assisted by a method called bimolecular fluorescent complementation (BiFC), which can detect weak and transient protein-protein interactions and reveal where the binding takes place in live cells, we have identified three effectors, Cdc42, CHMP6, and VPS4A that interact with Ras proteins in endosomes. These effectors are all necessary for Ras-induced transformation, suggesting that for Ras proteins to efficiently induce tumor formation, they must also activate effectors in cytoplasm, such as those in endosomes.

Regulation of Ras localization and cell transformation by evolutionarily conserved palmitoyltransferases.

Ras can act on the plasma membrane (PM) to mediate extracellular signaling and tumorigenesis. To identify key components controlling Ras PM localization, we performed an unbiased screen to seek Schizosaccharomyces pombe mutants with reduced PM Ras. Five mutants were found with mutations affecting the same gene, S.

Escorting Ras.

Ras proteins are best known to function on the plasma membrane to mediate growth factor signaling. Controlling the length of time that Ras proteins stay on the plasma membrane is an effective way to properly modulate the intensity and duration of growth factor signaling. It has been shown previously that H- and N-Ras proteins in the GTP-bound state can be ubiquitylated via a K-63 linkage, which leads to endosome internalization and results in a negative-feedback loop for efficient signal attenuation.

An intraoperative multimodal neurophysiologic approach to successful resection of precentral gyrus epileptogenic lesions.

Cortical dysplasias (CDs) are highly epileptogenic lesions with a good prognosis of seizure freedom, if totally resected. However, their accurate delineation and resection can be difficult, and depend on the extent of pathology and lesion location. Intraoperative neurophysiologic assessments are valuable in these situations.

Schizosaccharomyces pombe Arc3 is a conserved subunit of the Arp2/3 complex required for polarity, actin organization, and endocytosis.

We characterized the Schizosaccharomyces pombe arc3 gene, whose product shares sequence homology with that of the budding yeast ARC18 and human ARPC3/p21 subunits of the Arp2/3 complex. Our data showed that Arc3p co-localizes with F-actin patches at the cell ends, but not with F-actin cables or the equatorial actin ring, and binds other subunits of the Arp2/3 complex. Gene deletion analysis showed that arc3 is essential for viability.

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies.

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice.

Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe.

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome deficiencies; when its expression is repressed, proteasome deficiencies intensify.

Int6 and Moe1 interact with Cdc48 to regulate ERAD and proper chromosome segregation.

Int6/eIF3e is implicated in tumorigenesis, but its molecular functions remain unclear. We have studied its fission yeast homolog Yin6, reporting that it regulates proteolysis by controlling the assembly/localization of proteasomes, and binds directly to another conserved protein, Moe1. In the present study, we isolated Cdc48 as a Moe1-binding protein from a yeast two-hybrid screen, and confirmed biochemically that they form a stable complex in fission yeast.

The eIF3 interactome reveals the translasome, a supercomplex linking protein synthesis and degradation machineries.

eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome.

Isolation of the Schizosaccharomyces pombe proteasome subunit Rpn7 and a structure-function study of the proteasome-COP9-initiation factor domain.

Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome.

Spatial segregation of Ras signaling: new evidence from fission yeast.

The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question--how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments.

Compartmentalized signaling of Ras in fission yeast.

Compartment-specific Ras signaling is an emerging paradigm that may explain the multiplex outputs from a single GTPase. The fission yeast, Schizosaccharomyces pombe, affords a simple system in which to study Ras signaling because it has a single Ras protein, Ras1, that regulates two distinct pathways: one that controls mating through a Byr2-mitogen-activated protein kinase cascade and one that signals through Scd1-Cdc42 to maintain elongated cell morphology. We generated Ras1 mutants that are restricted to either the endomembrane or the plasma membrane.

ZOMES III: the interface between signalling and proteolysis. Meeting on The COP9 Signalosome, Proteasome and eIF3.

Meeting on The COP9 Signalosome, Proteasome and eIF3

Schizosaccharomyces pombe Ras1 effector, Scd1, interacts with Klp5 and Klp6 kinesins to mediate cytokinesis.

Fission yeast Scd1 is an exchange factor for Cdc42 and an effector of Ras1. In a screen for scd1 interacting genes, we isolated klp5 and klp6, which encode presumptive kinesins. Klp5 and Klp6 form a complex to control the same processes, which so far include microtubule dynamics and chromosome segregation.