Ribociclib induces broad chemotherapy resistance and EGFR dependency in ESR1 wildtype and mutant breast cancer

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15–40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatmen

Genome Sequence of Fusobacterium nucleatum Subspecies Polymorphum — a Genetically Tractable Fusobacterium

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common

Complement factor H, vitronectin, and opticin are tyrosine-sulfated proteins of the retinal pigment epithelium.

Lack of tyrosine sulfation of ocular proteins results in disorganized photoreceptor structure and drastically reduced visual function, demonstrating the importance of this post-translational modification to vision. To understand the role that tyrosine sulfation plays in the function of ocular proteins, we identified some tyrosine-sulfated proteins in the retinal pigment epithelium using two independent methods, immuno-affinity column purification with an anti-sulfotyrosine specific antibody and computer-based sequence analysis of retinal pigment epithelium secretome by means of the prediction program Sulfinator. Radioactive labeling followed by thin layer electrophoresis revealed that three proteins, vitronectin, opticin, and complement factor H (CFH), were post-translationally modified by tyrosine sulfation. The identification of vitronectin and CFH as tyrosine-sulfated proteins is significant, since both are deposited in drusen in the eyes of patients with age-related macular degeneration (AMD). Furthermore, mutations in CFH have been determined to be a major risk factor in the development of AMD. Future studies that seek to understand the role of CFH in the development of AMD should take into account the role that tyrosine sulfation plays in the interaction of this protein with its partners, and examine whether modulating sulfation provides a potential therapeutic target

PSG2-immunoaffinity column purification of tyrosine-sulfated proteins from cow RPE.

<p>(A). The elution profile was monitored by following absorbance at 280 nm. Following loading, the column was washed with buffers W1, W2, and W3. Elution was performed in buffer W3 containing 4 mM sulfated pentapeptide (EB). (B). Twenty-six microliter aliquots from input (IN), flow-through (FT), wash 1 (W1), and wash 2 (W2) were fractionated by SDS-PAGE, and proteins were visualized by staining with Coomassie blue dye. (C). Left, SDS-PAGE of 26 µL of wash 3 (W3) and eluted samples (EB) from the immunoaffinity column stained with Coomassie blue (CB) and right, immunoblotted with PSG2. Asterisks indicate the bands that were prominent on Coomassie blue-stained gel (CB) and were also recognized by PSG2 as tyrosine-sulfated.</p

Native human RPE vitronectin is tyrosine-sulfated.

<p>Vitronectin was immunoprecipitated from 500 µg human RPE lysates using anti-VTN antibody (lane 2) or mouse IgG (lane 3). Immunoprecipitants were fractionated by SDS-PAGE, transferred, and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times using biologically different human RPE samples. (B). Ectopically-expressed vitronectin is tyrosine-sulfated. Recombinant VTN or empty vector (pcDNA3.1) were transfected into HEK 293T cells and immunoprecipitated from conditioned media using anti-VTN antibody (lane 4) or mouse IgG (lane 3). Immunoprecipitants were electrophoresed and immunoblotted using anti-VTN antibody or anti-sulfotyrosine PSG2 antibody. Immunoprecipitation and western blots were repeated 3 independent times after independent VTN transfections. (C). ³⁵S-metabolic labeling of recombinant vitronectin <i>in vitro</i>. Vitronectin-transfectants were radiolabelled with ³⁵Sulfate. Following radiolabeling, vitronectin was immunoprecipitated and blots were either subjected to autoradiography (AR) or immunoblotted with anti-VTN antibody. (D). Radiolabeled vitronectin bands were excised from the membrane along with equivalent areas from mouse IgG immunoprecipitants, and alkaline hydrolysis was performed. The samples were then spiked with sulfo-amino standards tyrosine sulfate, threonine sulfate, and serine sulfate, and subjected to thin layer electrophoresis (TLE) on cellulose plates. Following TLE analysis, sulfo-amino standards (NHD) were visualized either by spraying with Ninhydrin or autoradiography (AR). TLE experiments were repeated at least three independent times.</p

List of potential tyrosines that may be sulfated on human and cow vitronectin, opticin, and CFH, as identified by Sulfinator.

<p>Those sites that have been experimentally proven are marked by an asterisks and citation is provided.</p

List of potential tyrosine-sulfated proteins in cow RPE.

<p>Sixteen proteins were identified in cow RPE by MALDI-MS analysis of SDS-PAGE gel slices of PSG2 immunoaffinity column eluent. The asterisks mark those proteins at 75 and 50 kD, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105409#pone-0105409-g002" target="_blank">Figure 2C</a>. NCBI reference sequence database ID numbers (RefSeq#) and percent sequence coverage (coverage %) are also indicated for each protein. Inclusion in this table does not confirm that the protein is tyrosine-sulfated. Some of the proteins may be isolated by the affinity column as a result of their interaction with sulfated proteins.</p

Immunoblot analysis of neurosensory (NS) retinal and RPE extracts to identify tyrosine-sulfated proteins.

<p>Immunoblot analysis of 50 µg of RPE (lanes 1–4) and neurosensory retinal extracts (lanes 5–8) from human, cow, pig, and mouse RPE probed with the anti-sulfotyrosine antibody PSG2. Blots were repeated 3 independent times using biologically different samples.</p