A quick pipeline for the isolation of 3D cell culture-derived extracellular vesicles.

Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture-derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time-dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC-based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells.

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

Violacein negatively modulates the colorectal cancer survival and epithelial-mesenchymal transition.

Violacein is a secondary metabolite produced by several microorganisms including Chromobacterium violaceum, and it is already used in food and cosmetics. However, due to its potent anticancer and low side effects, its molecular action needs to be deeply scrutinized. Therefore, the main objective of this study was to evaluate the violacein's ability to interfere with three cancer hallmarks: growth factors receptor-dependent signaling, proliferation, and epithelial-mesenchymal transition (EMT). Violacein has been associated with the induction of apoptosis in colorectal cancer (CRC) cells. Here, we demonstrate that this molecule is also active in CRC spheroids and inhibits cell migration. Violacein treatment reduced the amount of EGFR and AXL receptors in the HT29 cell line. Accordingly, the inhibition of the AKT, ERK, and PKCδ kinases, which are downstream mediators of the signaling pathways triggered by EGFR and AXL, is detected. Another interesting finding was that even when the cells were stimulated with transforming growth factor-ß, the EMT marker (N-cadherin) decreased. Therefore, this study provides further evidence that reinforces the potential of violacein as an antitumor agent, once this biomolecule can "switch off" properties associated with cancer plasticity.

Kinome profiling of cholangiocarcinoma organoids reveals potential druggable targets that hold promise for treatment stratification.

BACKGROUND: Cholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma. METHODS: Kinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids. RESULTS: Kinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRß) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs. CONCLUSIONS: In conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine.

Platelet-dependent signaling and Low Molecular Weight Protein Tyrosine Phosphatase expression promote aggressive phenotypic changes in gastrointestinal cancer cells.

Over the last decades, some members of the protein tyrosine phosphatase family have emerged as cancer promoters. Among them, the Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) has been described to be associated with colorectal cancer liver metastasis and poor prostate cancer prognosis. Of importance in the process of cancer progression and metastasis is the interaction between tumor cells and platelets, as the latter are thought to promote several tumor hallmarks. Here, we examine to what extent LMWPTP expression in tumor cells affects their interaction with platelets. We demonstrate that the gene encoding LMWPTP is overexpressed in upper gastrointestinal (GI) cancer cell as well as colorectal cancer, and subsequently employ cell line models to show that the level of this phosphatase may be further augmented in the presence of platelets. We demonstrate that tumor-platelet interaction promotes GI tumor cell proliferation. Additionally, using know-down/-out models we show that LMWPTP expression in cancer cells contributes to a more efficient interaction with platelets and drives platelet-induced proliferation. These data are the first to demonstrate that phosphatases play a positive role in the tumor-promoting activities of platelets, with LMWPTP emerging as a key player promoting oncogenic phenotypic changes in tumor cells.

The role of phospho-tyrosine signaling in platelet biology and hemostasis.

Platelets are small enucleated cell fragments specialized in the control of hemostasis, but also playing a role in angiogenesis, inflammation and immunity. This plasticity demands a broad range of physiological processes. Platelet functions are mediated through a variety of receptors, the concerted action of which must be tightly regulated, in order to allow specific and timely responses to different stimuli. Protein phosphorylation is one of the main key regulatory mechanisms by which extracellular signals are conveyed. Despite the importance of platelets in health and disease, the molecular pathways underlying the activation of these cells are still under investigation. Here, we review current literature on signaling platelet biology and in particular emphasize the newly emerging role of phosphatases in these processes.

Low molecular weight protein tyrosine phosphatase as signaling hub of cancer hallmarks.

In the past decade, significant progress has been made in understanding the role of protein tyrosine phosphatase as a positive regulator of tumor progression. In this scenario, our group was one of the first to report the involvement of the low molecular weight protein tyrosine phosphatase (LMWPTP or ACP1) in the process of resistance and migration of tumor cells. Later, we and others demonstrated a positive correlation between the amount of this enzyme in human tumors and the poor prognosis. With this information in mind, we asked if LMWPTP contribution to metastasis, would it have an action beyond the primary tumor site. We know that the amount of this enzyme in the tumor cell correlates positively with the ability of cancer cells to interact with platelets, an indication that this enzyme is also important for the survival of these cells in the bloodstream. Here, we discuss several molecular aspects that support the idea of LMWPTP as a signaling hub of cancer hallmarks. Chemical and genetic modulation of LMWPTP proved to shut down signaling pathways associated with cancer aggressiveness. Therefore, advances in the development of LMWPTP inhibitors have great applicability in human diseases such as cancer.

Platelets in aging and cancer-"double-edged sword".

Platelets control hemostasis and play a key role in inflammation and immunity. However, platelet function may change during aging, and a role for these versatile cells in many age-related pathological processes is emerging. In addition to a well-known role in cardiovascular disease, platelet activity is now thought to contribute to cancer cell metastasis and tumor-associated venous thromboembolism (VTE) development. Worldwide, the great majority of all patients with cardiovascular disease and some with cancer receive anti-platelet therapy to reduce the risk of thrombosis. However, not only do thrombotic diseases remain a leading cause of morbidity and mortality, cancer, especially metastasis, is still the second cause of death worldwide. Understanding how platelets change during aging and how they may contribute to aging-related diseases such as cancer may contribute to steps taken along the road towards a "healthy aging" strategy. Here, we review the changes that occur in platelets during aging, and investigate how these versatile blood components contribute to cancer progression.

Correction: Faria, A.V.S. et al., Targeting Tyrosine Phosphatases by 3-Bromopyruvate Overcomes Hyperactivation of Platelets from Gastrointestinal Cancer Patients. J. Clin. Med. 2019, 8, 936.

The authors wish to make the following correction to their paper [...].

LMWPTP modulates the antioxidant response and autophagy process in human chronic myeloid leukemia cells.

In the last decade, several reports highlight the importance of the low molecular weight protein tyrosine phosphatase (LMWPTP) in cancer aggressiveness and resistance. Specifically, in chronic myeloid leukemia, we have reported that high expression of the LMWPTP maintains Src and Bcr-Abl kinases in an activated status and the glucose metabolism is directed to lactate production and, in turn, favor the pentoses pathway (one of the key process for antioxidant and protective responses). In this present study, we investigated the possible correlation between the LMWPTP and autophagy. In resistant chronic myeloid leukemia cells, the antioxidant response is supported by the glycolytic metabolism and antioxidant enzymes such as SOD and catalase, both favored by the LMWPTP. Therefore, when the cells were challenged by hydrogen peroxide treatment, the LMWPTP level goes down as well as SOD, and in turn, autophagy process was stimulated. The findings presented here reveal a novel aspect by which LMWPTP cooperates for the resistance of CML towards stressor stimuli.

Targeting Tyrosine Phosphatases by 3-Bromopyruvate Overcomes Hyperactivation of Platelets from Gastrointestinal Cancer Patients.

Venous thromboembolism (VTE) is one of the most common causes of cancer related mortality. It has been speculated that hypercoagulation in cancer patients is triggered by direct or indirect contact of platelets with tumor cells, however the underlying molecular mechanisms involved are currently unknown. Unraveling these mechanisms may provide potential avenues for preventing platelet-tumor cell aggregation. Here, we investigated the role of protein tyrosine phosphatases in the functionality of platelets in both healthy individuals and patients with gastrointestinal cancer, and determined their use as a target to inhibit platelet hyperactivity. This is the first study to demonstrate that platelet agonists selectively activate low molecular weight protein tyrosine phosphatase (LMWPTP) and PTP1B, resulting in activation of Src, a tyrosine kinase known to contribute to several platelet functions. Furthermore, we demonstrate that these phosphatases are a target for 3-bromopyruvate (3-BP), a lactic acid analog currently investigated for its use in the treatment of various metabolic tumors. Our data indicate that 3-BP reduces Src activity, platelet aggregation, expression of platelet activation makers and platelet-tumor cell interaction. Thus, in addition to its anti-carcinogenic effects, 3-BP may also be effective in preventing platelet-tumor cell aggregationin cancer patients and therefore may reduce cancer mortality by limiting VTE in patients.

Oncophosphosignaling Favors a Glycolytic Phenotype in Human Drug Resistant Leukemia.

In chemoresistant leukemia cells (Lucena-1), the low molecular weight protein tyrosine phosphatase (LMWPTP) is about 20-fold more active than in their susceptible counterpart (K562). We found this phosphatase ensures the activated statuses of Src and Bcr-Abl. Since, phosphorylation and dephosphorylation of proteins represent a key post-translational regulation of several enzymes, we also explored the kinome. We hereby show that LMWPTP superactivation, together with kinome reprogramming, cooperate towards glucose addiction. Resistant leukemia cells present lower levels of oxidative metabolism, in part due to downexpression of the following mitochondrial proteins: pyruvate dehydrogenase subunit alpha 1, succinate dehydrogenase, and voltage-dependent anion channel. Those cells displayed higher expression levels of glucose transporter 1 and higher production of lactate. In addition, Lucena-1 siRNA LMWPTP cells showed lower expression levels of glucose transporter 1 and lower activity of lactate dehydrogenase. On the other hand, K562 cells overexpressing LMWPTP presented higher expression/activity of both proteins. In this study, we show that LMWPTP is a pivotal mediator of metabolic reprogramming that confers survival advantages to leukemia cells against death stimuli. J. Cell. Biochem. 118: 3846-3854, 2017. © 2017 Wiley Periodicals, Inc.