High p16 expression and heterozygous RB1 loss are biomarkers for CDK4/6 inhibitor resistance in ER breast cancer.

CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89).

Inactivating Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.

Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity. A panel of cell lines representative of TNBC was tested for and sensitivity to CDK4/6 inhibition.

High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial.

Unlabelled: FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control.

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs.

Functional Genetic Screen Identifies Increased Sensitivity to WEE1 Inhibition in Cells with Defects in Fanconi Anemia and HR Pathways.

WEE1 kinase regulates CDK1 and CDK2 activity to facilitate DNA replication during S-phase and to prevent unscheduled entry into mitosis. WEE1 inhibitors synergize with DNA-damaging agents that arrest cells in S-phase by triggering direct mitotic entry without completing DNA synthesis, resulting in catastrophic chromosome fragmentation and apoptosis. Here, we investigated how WEE1 inhibition could be best exploited for cancer therapy by performing a functional genetic screen to identify novel determinants of sensitivity to WEE1 inhibition.

Parallel RNA interference screens identify EGFR activation as an escape mechanism in FGFR3-mutant cancer.

Unlabelled: Activation of fibroblast growth factor receptors (FGFR) is a common oncogenic event. Little is known about the determinants of sensitivity to FGFR inhibition and how these may vary between different oncogenic FGFRs. Using parallel RNA interference (RNAi) genetic screens, we show that the EGF receptor (EGFR) limits sensitivity to FGFR inhibition in FGFR3-mutant and -translocated cell lines, but not in other FGFR-driven cell lines.

Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration.

Background: Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways.

Results: Here we use data from a genome-wide RNAi morphology screen in Drosophila melanogaster cells together with bioinformatics to identify 26 new regulators of morphology and cytoskeletal organization in human cells.

FGFR signaling promotes the growth of triple-negative and basal-like breast cancer cell lines both in vitro and in vivo.

Purpose: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation.

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential.

Protein-tyrosine phosphatase-alpha and Src functionally link focal adhesions to the endoplasmic reticulum to mediate interleukin-1-induced Ca2+ signaling.

Calcium (Ca2+) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) alpha in regulating IL-1-induced Ca2+ signaling in fibroblasts. IL-1 promoted recruitment of PTPalpha to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca2+ release channel IP3R.

Experimental ventilator-induced lung injury: exacerbation by positive end-expiratory pressure.

Background: Previous experimental studies of ventilator-induced lung injury have shown that positive end-expiratory pressure (PEEP) is protective. The authors hypothesized that the application of PEEP during volume-controlled ventilation with a moderately high tidal volume (VT) in previously healthy in vivo rats does not attenuate ventilator-induced lung injury if the peak airway pressure markedly increases during the application of PEEP.

Methods: Sixty healthy, male Sprague-Dawley rats were anesthetized and randomized to be mechanically ventilated for 4 h at (1) VT of 6 ml/kg, (2) VT of 20 ml/kg, or (3) VT of 20 ml/kg plus 10 cm H2O of PEEP.

Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein.

Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear.

Tyrosine phosphatase PTPalpha regulates focal adhesion remodeling through Rac1 activation.

We characterized the role of protein tyrosine phosphatase (PTP)-alpha in focal adhesion (FA) formation and remodeling using wild-type and PTPalpha-deficient (PTPalpha(-/-)) cells. Compared with wild-type cells, spreading PTPalpha(-/-) fibroblasts displayed fewer leading edges and formed elongated alpha-actinin-enriched FA at the cell periphery. These features suggest the presence of slowly remodeling cell adhesions and were phenocopied in human fibroblasts in which PTPalpha was knocked down using short interfering RNA (siRNA) or in NIH-3T3 fibroblasts expressing catalytically inactive (C433S/C723S) PTPalpha.

Phosphorylation of SHP-2 regulates interactions between the endoplasmic reticulum and focal adhesions to restrict interleukin-1-induced Ca2+ signaling.

Interleukin-1 (IL-1)-induced Ca2+ signaling in fibroblasts is constrained by focal adhesions. This process involves the proteintyrosine phosphatase SHP-2, which is critical for IL-1-induced phosphorylation of phospholipase Cgamma1, thereby enhancing IL-1-induced Ca2+ release and ERK activation. Currently, the mechanisms by which SHP-2 modulates Ca2+ release from the endoplasmic reticulum are not defined.

Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions.

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling.

SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1.

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2.

The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts.

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation.