The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases

SHP-1 expression and localization during hypoxia.

<p>(A) The induction of HIF-1α protein levels by 1h and 4h hypoxia was confirmed by western blot (n = 4). (B) HIF-1α (green) and SHP-1 (red) were detected by immunofluorescence staining. DAPI (blue) was used to visualize nuclei. HIF-1α was induced by hypoxia (***p<0.001; n = 6). SHP-1 expression was also slightly induced by hypoxia (p<0.05; n = 6). Both SHP-1 and HIF-1α were shown to be predominantly located in the nucleus with moderate expression in the cytoplasm. Graphs next to photos show fluorescent intensities of SHP-1 and HIF-1α (n = 6 of 6 visual fields/sample) (C) Immunoprecipitation was performed for SHP-1. HIF-1α could be detected in precipitates of SHP-1 (n = 6). IgG: IgG isotype control antibody. (D) HIF-1α could successfully be immunoprecipitated by a specific antibody (lane 6 upper blot) and is tyrosine phosphorylated under normoxia and hypoxia as seen in MG132 (10μM) treated cells (lane 3 and 4 lower blot, n = 3). IgG: IgG isotype control antibody.</p

SHP-1 knock-down enhances ROS production during hypoxia.

<p>(A) SHP-1 knock-down enhanced ROS formation during hypoxia (*p<0.001; n = 12) compared to control AS-Odn treated cells. (B) SHP-1 knock-down enhanced hypoxia induced intracellular superoxide production (*p<0.05, n = 8), as assessed by DHE fluorescence.</p

SHP-1 destabilizes HIF-1α protein during hypoxia.

<p>(A) HIF-1α protein levels were further enhanced by SHP-1 knock-down during hypoxia (4h) (*p<0.05, n = 4, lane 4) compared to control conditions (lane 3). Graph shows the protein band density of HIF-1α in relation to loading control β-Actin. (B) HIF-1α levels in non transfected cells was compared to SHP-1 WT expressing cells showing no influence of transfection on this (upper blot; n = 4). HMECs expressing inactive SHP-1 (CS) showed increased (*p<0.05; n = 8, lane 2 in lower blot), whereas expression of constitutively active SHP-1 (E74A) showed decreased (*p<0.05; n = 9, lane 3 in lower blot) levels of HIF-1α compared to wildtype (WT) SHP-1 (lane 1 in lower blot). Graph shows HIF-1α protein band density in relation to the loading control β-Actin. (C) HIF-1α mRNA was quantified by qRT-PCR. Hypoxia (4h) significantly decreased HIF-1α mRNA compared to normoxic conditions (*p<0.05; n = 10). However, SHP-1 knock-down had no effect on HIF-1α mRNA levels (n = 10). (D) The regulatory effect of SHP-1 on HIF-1α levels shown in (A) could not be observed when protein degradation was prevented by using the proteasome inhibitor MG132 (10μM) (n = 4). Graph shows HIF-1α protein band density in relation to the loading control β-Actin. (E) Differences in HIF-1α translation between cells expressing catalytic inactive (CS) and wildtype (WT) SHP-1 were not detected (n = 8) and the decreased HIF-1α accumulation seen in cells expressing constitutively active (E74A) SHP-1 was rescued upon inhibition of proteasomal inhibition (MG132 10μM) (n = 8).</p

Cathepsin S alterations induce a tumor-promoting immune microenvironment in follicular lymphoma

Bararia et al. discover and functionally characterize a clinically relevant mechanism of tumor and immune cell interaction in follicular lymphoma, a prototypical type of blood cancer. Cathepsin S alterations result in aberrant hyperactivity of this lysosomal cysteine protease and induce a tumor-promoting CD4 T cell enriched immune microenvironment.Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4 T cells in vitro. Tumors with hyperactive CTSS showed increased CD4 T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment

Distinct Hodgkin lymphoma subtypes defined by noninvasive genomic profiling.

The scarcity of malignant Hodgkin and Reed-Sternberg (HRS) cells hamper tissue-based comprehensive genomic profiling of classic Hodgkin lymphoma (cHL). Liquid biopsies, in contrast, show promise for molecular profiling of cHL due to relatively high circulating tumor DNA (ctDNA) levels. Here, we show that the plasma representation of mutations exceeds the bulk tumor representation in most cases, making cHL particularly amenable to noninvasive profiling. Leveraging single-cell transcriptional profiles of cHL tumors, we demonstrate HRS ctDNA shedding to be shaped by DNASE1L3, whose increased tumor microenvironment-derived expression drives high ctDNA concentrations. Using this insight, we comprehensively profile 366 patients, revealing two distinct cHL genomic subtypes with characteristic clinical and prognostic correlates, as well as distinct transcriptional and immunological profiles. Furthermore, we identify a novel class of truncating IL4R-mutations that are dependent on IL13 signaling and therapeutically targetable with IL4R blocking antibodies. Finally, using PhasED-Seq we demonstrate the clinical value of pre- and on-treatment ctDNA levels for longitudinally refining cHL risk prediction, and for detection of radiographically occult minimal residual disease. Collectively, these results support the utility of noninvasive strategies for genotyping and dynamic monitoring of cHL as well as capturing molecularly distinct subtypes with diagnostic, prognostic, and therapeutic potential