Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results:PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K-AKT-mTOR signaling network. Clin Cancer Res; 23(16); 4919-28. ©2017 AACR.

Genomic alterations in DNA repair and chromatin remodeling genes in estrogen receptor-positive metastatic breast cancer patients with exceptional responses to capecitabine.

We analyzed the genomic and phosphoproteomic profiles of breast cancer tissue obtained from six patients with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer who had highly durable (≥ 5 years) and, in some cases, ongoing clinical responses with capecitabine. Formalin-fixed, paraffin-embedded tissue samples from patients' primary (n = 4) or metastatic (n = 2) breast cancers were utilized for targeted next-generation sequencing and reversed phase protein microarray. Two patients received capecitabine monotherapy. Four patients received capecitabine in combination with paclitaxel; three of these continued single-agent capecitabine after stopping paclitaxel. Capecitabine was discontinued for progressive disease after a mean of 66 months in four patients (range 54-86 months), and two patients remain on therapy, having received capecitabine for >91 months and >122 months, respectively. Three patients' cancers (50%) had likely functional alterations in DNA repair and chromatin remodeling genes, while three other patients' cancers had variants of unknown significance in these pathways. Mutations in PIK3CA, amplifications of FGFR1 or ZNF703, or phosphorylation of HER family receptors and their downstream proteins did not preclude exceptional responses to capecitabine. None of the patients' tumors harbored TP53 or PTEN mutations. Four of the patients had breast cancer tissue available for PTEN immunohistochemistry, and all four patients' cancers were positive for PTEN. These surprising findings in a group of phenotypically similar patients with ER-positive, endocrine therapy-pretreated, HER2-negative metastases, are supported by preclinical data showing that sensitivity to 5-fluorouracil is enhanced by deficiencies in chromatin remodeling and homologous recombination genes. Our findings suggest that mutations that inactivate homologous recombination and/or chromatin remodeling genes within ER-positive, HER2-negative breast cancers may predict for highly durable responses to capecitabine.

A phase 1 study of cetuximab and lapatinib in patients with advanced solid tumor malignancies.

BACKGROUND: Acquired resistance to antiepidermal growth factor receptor (anti-EGFR) therapy may be caused by EGFR-v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ErbB2) heterodimerization and pathway reactivation. In preclinical studies, inhibiting ErbB2 blocked this resistance mechanism and resensitized cells to anti-EGFR therapy. Cetuximab targets EGFR, whereas lapatinib inhibits both EGFR and ErbB2. The objective of this phase 1 trial was to assess the safety, dose-limiting toxicities (DLTs), and maximum tolerated doses (MTDs) of cetuximab and lapatinib in patients with solid tumors. METHODS: Patients received standard weekly cetuximab with escalating lapatinib doses of 750 mg, 1000 mg, or 1250 mg daily in 3-week cycles. DLTs were monitored through the end of cycle 2. Pretreatment and post-treatment tumor biopsies and germline DNA samples were obtained for correlative studies. RESULTS: Twenty-two patients were enrolled, and 18 patients each were evaluable for toxicity and response. Fifty-nine percent of patients had received prior anti-EGFR therapy. Common toxicities included rash and diarrhea. No patient experienced a DLT at the highest dose level, and no grade 4 toxicity was observed. Response included no complete responses, 3 partial responses, 9 patients with stable disease, and 6 patients with disease progression, for an overall response rate of 17% and a clinical benefit rate of 67%. The clinical benefit rate in patients who had previously received anti-EGFR therapy was 70%. The mean treatment duration was 4.7 cycles (range, 1-14 cycles). Decreased expression of EGFR/ErbB2 pathway components after treatment was correlated with response, whereas increased expression in the PI3K, Jak/Stat, and MAPK pathways occurred in nonresponders. CONCLUSIONS: The combination of cetuximab and lapatinib was well tolerated, had the expected toxicities, and exhibited notable clinical activity, including in patients who had received previous anti-EGFR therapy. Further clinical study of this combination is warranted.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Transmembrane protein-free membranes fuse into xenopus nuclear envelope and promote assembly of functional pores.

Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5'-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly.

Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly.

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.

Transmembrane proteins are not required for early stages of nuclear envelope assembly.

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.

Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.

Reversible stages of the low-pH-triggered conformational change in influenza virus hemagglutinin.

The refolding of the prototypic fusogenic protein hemagglutinin (HA) at the pH of fusion is considered to be a concerted and irreversible discharge of a loaded spring, with no distinct intermediates between the initial and final conformations. Here, we show that HA refolding involves reversible conformations with a lifetime of minutes. After reneutralization, low pH-activated HA returns from the conformations wherein both the fusion peptide and the kinked loop of the HA2 subunit are exposed, but the HA1 subunits have not yet dissociated, to a structure indistinguishable from the initial one in functional, biochemical and immunological characteristics. The rate of the transition from reversible conformations to irreversible refolding depends on the pH and on the presence of target membrane. Importantly, recovery of the initial conformation is blocked by the interactions between adjacent HA trimers. The existence of the identified reversible stage of refolding can be crucial for allowing multiple copies of HA to synchronize their release of conformational energy, as required for fusion.

Spontaneous lipid vesicle fusion with electropermeabilized cells.

Fusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process.

Cell hybridization by electrofusion on filters.

Electric field pulses induce permeabilization and associated fusogenicity in cell membranes. Electrofusion of cells is usually performed in two steps: the first is the creation of close intercellular contacts; the second is an application of electric pulses that induces membrane fusion. Very large cell contacts can be obtained by a filter aspiration method. A cell monolayer is created by controlled suction on biocompatible filter. No spontaneous fusion results. Just after filtration, electrofusion is obtained by field pulses applied parallel to the filter. Cell viability is not strongly affected and cells recover their spherical shape in the minute time range after filtration. The electrical parameters, the cell density, and the flow rate control fusion. Fusion is obtained with cells of different origins with very different adhesion properties. Hybrid cells are easily formed. This approach appears to be a very efficient method for cell hybridization with an easy-to-use protocol.

Mechanism of isoniazid uptake in Mycobacterium tuberculosis.

Initial transport kinetics of isoniazid (INH) and its uptake at the plateau were studied in Mycobacterium tuberculosis H37Rv under various experimental conditions. The initial uptake velocity increased linearly with INH concentration from 2 x 10(-6) M to 10(-2) M. It was modified neither by addition of a protonophore that abolished proline transport, nor following ATP depletion by arsenate, which inhibited glycerol uptake, two transport processes taken as controls for secondary active transport and facilitated diffusion, respectively. Microaerobiosis or low temperature (4 degrees C) were without effect on initial uptake. It is thus likely that INH transport in M. tuberculosis proceeds by a passive diffusion mechanism, and that catalase-peroxidase (KatG) is not involved in the actual transport. However, conditions inhibiting KatG activity (high INH concentration, microaerobiosis, low temperature) decrease cell radioactivity at the uptake plateau. It is proposed that INH transport occurs by passive diffusion. KatG is involved only in the intracellular accumulation of oxidized derivatives of INH, especially of isonicotinic acid, which is trapped inside cells in its ionized form. This model explains observed and previously known characteristics of the accumulation of radioactivity in the presence of [14C]INH for various species and strains of mycobacteria.