Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells.

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529-37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869-84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

Phenotypic plasticity during metastatic colonization.

Most solid cancer-related deaths result from metastasis, a multistep process in which cancer cells exit the primary site, intravasate into the bloodstream, extravasate, and colonize distant organs. Colonization is facilitated by clonal selection and the high phenotypic plasticity of cancer cells that creates reversible switching of cellular states. Cancer cell plasticity leads to intratumor heterogeneity and fitness, yielding cells with molecular and cellular programs that facilitate survival and colonization. While cancer cell plasticity is sometimes limited to the process of epithelial-to-mesenchymal transition (EMT), recent studies have broadened its definition. Plasticity arises from both cell-intrinsic and cell-extrinsic factors and is a major obstacle to efficacious anti-cancer therapies. Here, we discuss the multifaceted notion of cancer cell plasticity associated with metastatic colonization.

Sphingomyelin Biosynthesis Is Essential for Phagocytic Signaling during Mycobacterium tuberculosis Host Cell Entry.

Phagocytosis by alveolar macrophages is the obligate first step in Mycobacterium tuberculosis (Mtb) infection, yet the mechanism underlying this process is incompletely understood. Here, we show that Mtb invasion relies on an intact sphingolipid biosynthetic pathway. Inhibition or knockout of early sphingolipid biosynthetic enzymes greatly reduces Mtb uptake across multiple phagocytic cell types without affecting other forms of endocytosis. While the phagocytic receptor dectin-1 undergoes normal clustering at the pathogen contact sites, sphingolipid biosynthetic mutant cells fail to segregate the regulatory phosphatase CD45 from the clustered receptors. Blocking sphingolipid production also impairs downstream activation of Rho GTPases, actin dynamics, and phosphoinositide turnover at the nascent phagocytic cup. Moreover, we found that production of sphingomyelin, not glycosphingolipids, is essential for Mtb uptake. Collectively, our data support a critical role of sphingomyelin biosynthesis in an early stage of Mtb infection and provide novel insights into the mechanism underlying phagocytic entry of this pathogen.IMPORTANCEMycobacterium tuberculosis (Mtb) invades alveolar macrophages through phagocytosis to establish infection and cause disease. The molecular mechanisms underlying Mtb entry are still poorly understood. Here, we report that an intact sphingolipid biosynthetic pathway is essential for the uptake of Mtb by phagocytes. Disrupting sphingolipid production affects the segregation of the regulatory phosphatase CD45 from the nascent phagosome, a critical step in the progression of phagocytosis. We also show that blocking sphingolipid biosynthesis impairs activation of small GTPases and phosphoinositide turnover at the host-pathogen contact sites. Moreover, production of sphingomyelin, not glycosphingolipids, is critical for the phagocytic uptake of Mtb These data demonstrate a vital role for sphingomyelin biosynthesis in an early step of Mtb infection, defining a potential target for antimycobacterial therapeutics.

Visualizing the Early Stages of Phagocytosis.

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system - such as neutrophils, dendritic cells, and macrophages - retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4. Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 µm, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

A Global Analysis of CYP51 Diversity and Azole Sensitivity in Rhynchosporium commune.

CYP51 encodes the target site of the azole class of fungicides widely used in plant protection. Some ascomycete pathogens carry two CYP51 paralogs called CYP51A and CYP51B. A recent analysis of CYP51 sequences in 14 European isolates of the barley scald pathogen Rhynchosporium commune revealed three CYP51 paralogs, CYP51A, CYP51B, and a pseudogene called CYP51A-p. The same analysis showed that CYP51A exhibits a presence/absence polymorphism, with lower sensitivity to azole fungicides associated with the presence of a functional CYP51A. We analyzed a global collection of nearly 400 R. commune isolates to determine if these findings could be extended beyond Europe. Our results strongly support the hypothesis that CYP51A played a key role in the emergence of azole resistance globally and provide new evidence that the CYP51A gene in R. commune has further evolved, presumably in response to azole exposure. We also present evidence for recent long-distance movement of evolved CYP51A alleles, highlighting the risk associated with movement of fungicide resistance alleles among international trading partners.