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Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3–KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3–KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling. The multidomain scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) can bind GTP-bound Ras and Rap small GTPases. Here the authors show that, by binding active KRAS, SHANK3 maintains oncogenic KRAS/MAPK/ERK signaling at an optimal level while its depletion in KRAS-mutant cancer cell lines results in ERK signalling overdose and impaired cell proliferation. [ABSTRACT FROM AUTHOR]

In breast cancer, the currently approved anti‐receptor tyrosine‐protein kinase erbB‐2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2‐related therapeutic targets could offer a means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin‐related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2‐targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti‐SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cell density in a mouse xenograft model of HER2‐positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient‐derived explant three‐dimensional cultures. These results provide, for the first time, proof of principle that SorLA is a druggable target in breast cancer. [ABSTRACT FROM AUTHOR]

Author(s): Pia Rantakari [1]; Norma Jppinen [1]; Emmi Lokka [1]; Elias Mokkala [1]; Heidi Gerke [1]; Emilia Peuhu [2]; Johanna Ivaska [2, 3]; Kati Elima [1, 4]; Kaisa Auvinen [1]; [...]

Abstract Background cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells. Methods 5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone-associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student's t-test (p < 0.05). Results Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases. Conclusions Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma.

The article presents a study which identified insulin-like growth factor-binding protein 2 (IGFBP2) as a potent adipocrine factor secreted by healthy breast adipocytes that acts as a barrier against invasive progression. Topics discussed include reduction of breast cancer invasion by human umbilical vein endothelial cells (HUVEC)-derived angiocrine factor(s), potent anti-invasive effect of IGFBP2 secretion by HUVECs and fibroblasts, and flow cytometric analysis of IGFBP2 cell surface binding.

Routine histochemical techniques are capable of producing vast amount of information from diverse samples types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms., (© 2024. Published by The Company of Biologists Ltd.)

Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of α v β 3 -selective cyclic, dimeric peptide 68 Ga-DOTA-E[c(RGDfK)] 2 , where E[c(RGDfk)] 2 = glutamic acid-[cyclo(arginyl-glycyl-aspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice ( Sharpin cpdm , where cpdm = chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods: Sharpin cpdm mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide 68 Ga-DOTA-E[c(RGDfK)] 2 After the last 68 Ga-DOTA-E[c(RGDfK)] 2 peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of 68 Ga-DOTA-E[c(RGDfK)] 2 in the mouse skin and tumor was significantly higher in Sharpin cpdm mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with 68 Ga-DOTA-E[c(RGDfK)] 2 peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpin cpdm mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpin cpdm mice. Conclusion: Sharpin cpdm mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)