Decorin (DCN) Downregulation Activates Breast Stromal Fibroblasts and Promotes Their Pro-Carcinogenic Effects through the IL-6/STAT3/AUF1 Signaling.

Decorin (DCN), a member of the small leucine-rich proteoglycan gene family, is secreted from stromal fibroblasts with non-cell-autonomous anti-breast-cancer effects. Therefore, in the present study, we sought to elucidate the function of decorin in breast stromal fibroblasts (BSFs). We first showed DCN downregulation in active cancer-associated fibroblasts (CAFs) compared to their adjacent tumor counterpart fibroblasts at both the mRNA and protein levels. Interestingly, breast cancer cells and the recombinant IL-6 protein, both known to activate fibroblasts in vitro, downregulated DCN in BSFs. Moreover, specific DCN knockdown in breast fibroblasts modulated the expression/secretion of several CAF biomarkers and cancer-promoting proteins (α-SMA, FAP- α, SDF-1 and IL-6) and enhanced the invasion/proliferation abilities of these cells through activation of the STAT3/AUF1 signaling. Furthermore, DCN-deficient fibroblasts promoted the epithelial-to-mesenchymal transition and stemness processes in BC cells in a paracrine manner, which increased their resistance to cisplatin. These DCN-deficient fibroblasts also enhanced angiogenesis and orthotopic tumor growth in mice in a paracrine manner. On the other hand, ectopic expression of DCN in CAFs suppressed their active features and their paracrine pro-carcinogenic effects. Together, the present findings indicate that endogenous DCN suppresses the pro-carcinogenic and pro-metastatic effects of breast stromal fibroblasts.

Ovarian tumor cell-derived JAGGED2 promotes omental metastasis through stimulating the Notch signaling pathway in the mesothelial cells.

The primary site of metastasis for epithelial ovarian cancer (EOC) is the peritoneum, and it occurs through a multistep process that begins with adhesive contacts between cancer cells and mesothelial cells. Despite evidence that Notch signaling has a role in ovarian cancer, it is unclear how exactly it contributes to ovarian cancer omental metastasis, as well as the cellular dynamics and intrinsic pathways that drive this tropism. Here we show that tumor cells produced the Notch ligand Jagged2 is a clinically and functionally critical mediator of ovarian cancer omental metastasis by activating the Notch signaling in single-layered omental mesothelial cells. In turn, Jagged2 promotes tumor growth and therapeutic resistance by stimulating IL-6 release from mesothelial cells. Additionally, Jagged2 is a potent downstream mediator of the omental metastasis cytokine TGF-ß that is released during omental destruction. Importantly, therapeutic inhibition of Jagged2-mediated omental metastasis was significantly improved by directly disrupting the Notch pathway in omental mesothelial cells. These findings highlight the key role of Jagged2 to the functional interplay between the TGF-ß and the Notch signaling pathways during the metastatic process of ovarian cancer cells to the omentum and identify the Notch signaling molecule as a precision therapeutic target for ovarian cancer metastasis.

Ionizing radiation normalizes the features of active breast cancer stromal fibroblasts and suppresses their paracrine pro-carcinogenic effects.

BACKGROUND: Radiotherapy is an important therapeutic strategy for breast cancer patients through reducing the chances of recurrence and metastasis, which are fueled by cancer-associated fibroblasts (CAFs). Thereby, we addressed here the effect of various doses of X-rays on breast CAFs and their adjacent counterparts. METHODS: We have used WST1 and annexin V-associated with flow cytometry to test the cytotoxic effects of X-rays. Immunoblotting and ELISA was used to assess the expression/secretion of various proteins. Immunohistochemistry was utilized to determine the level of ß-galactosidase and Ki-67. Sphere formation assay was used to test the ability of breast cancer cells to form tumorspheres. Orthotopic tumor xenografts were also used to evaluate the effect of X-ray-treated breast stromal fibroblasts on breast cancer tumor growth in vivo. RESULTS: Breast stromal fibroblasts showed high resistance to X-rays. While the low dose (5 Gy) inhibited cell proliferation and the active features of CAFs, the higher doses (16 and 50 Gy) promoted senescence. However, this was not accompanied by the senescence-associated secretory phenotype (SASP), but rather a reduction in the synthesis/secretion of various cancer-associated cytokines. Additionally, X-rays suppressed the features of active breast stromal fibroblasts, and their paracrine pro-carcinogenic effects. The ablative dose (16 Gy) inhibited the capacity of active stromal fibroblasts to promote the pro-metastatic processes epithelial-to-mesenchymal transition, the formation of cancer stem cells, as well as the growth of humanized orthotopic breast tumor xenografts. CONCLUSION: Together, these findings indicate that X-rays can normalize the features of active breast stromal fibroblasts through promoting senescence without SASP.

Sheep as a Model for Liver Transplantation.

OBJECTIVE: Experimental animal liver transplantation is the initial step, before the application of the procedure on humans. Canine and swine transplantation were used to perfect the technical aspects of the procedure. Small animals such as rats were mainly utilized to study the metabolic and immunological aspects of liver transplantation. In this paper, we describe our experience with attempting liver transplantation in a sheep animal model. MATERIAL AND METHOD: The animal model used for both donor and recipient was outbred male weanling sheep of Naimi strain (Ovis aries, Awassi). They weigh between 25 and 35 kg. They were put under general anesthesia. Harvested livers were kept in cold storage. Recipients underwent hepatectomy, after construction of an active portal systemic bypass using a Medtronic pump. The implantation was done with caval replacement and direct portal anastomosis. The hepatic artery with its attachments to the aortal was anastomosed directly to the recipient aorta. RESULT: Twelve pairs (24 sheep) were utilized for donor and recipient surgery. Donor surgery was completed successfully in all 12 cases. Recipient surgery was not completed in three cases, when animals were lost in the implantation phase, before reperfusion mainly due to uncontrolled bleeding, resulting in hemodynamic instability. We also lost five recipients immediately after reperfusion, mainly due to post-perfusion bleeding and hemodynamic instability. Four recipients stayed alive after the implantation. CONCLUSION: We demonstrated the feasibility of using sheep as an animal model for liver transplantation. We described the similarities of sheep liver to that of humans, as well as the technical difficulties. This model is suitable in situations where other well-established models are not available for cultural or religious reasons. Further refinement in the technical aspects will be needed, as well as investigation of the biochemical outcome and long-term survival.

The use of ex-vivo liver perfusion circuit in sheep model: Preliminary report.

OBJECTIVES: To describe a novel animal model for ex-vivo liver perfusion. METHODS: This study was carried out at King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, between September 2016 and January 2019. We assembled a perfusion circuit operated by a continuous pressure-driven arterial pump with continuous portal and arterial pressure and volume measurements. We used normothermic oxygenated perfusate. The livers used were retrieved from the sheep. RESULTS: Ex-vivo continuous perfusion of the liver was achieved for up to 9 hours with stable pressure and volume in both hepatic artery and portal vein. In 4 experiments the arterial pressure was kept in a range of 48-52 mmHg with a mean of 51.75±4.31 resulting in arterial volume at steady state of 223.5±48.25 ml/minute (95% confidence level). At steady state the mean portal pressure was 16.25±1.45 mmHg with a mean volume of 854±313.75 ml/minute (95% confidence level). Bile production was observed during the perfusion period. Hemodynamic parameters were similar to the physiological parameters observed in normothermic perfusion model of the porcine liver. CONCLUSION: A normothermic oxygenated ex-vivo perfusion circuit was successfully constructed using the sheep liver. A sustainable functional circuit with physiological hemodynamic parameters was achieved. Further study on sheep model seems to be feasible.

Tocilizumab suppresses the pro-carcinogenic effects of breast cancer-associated fibroblasts through inhibition of the STAT3/AUF1 pathway.

Active breast cancer-associated fibroblasts (CAFs), the most influential cells in breast tumor microenvironment, express/secrete high levels of the proinvasive/metastatic interleukin-6 (IL-6). Therefore, we have tested here the effect of the IL-6 receptor (IL-6R) inhibitor tocilizumab (TCZ; Actemra) on different active breast CAFs. We have shown that TCZ potently and persistently suppresses the expression of various CAF biomarkers, namely α-SMA, SDF-1 as well as the STAT3 pathway and its downstream target AUF1. TCZ also inhibited the proliferation, migration and invasion abilities of active breast CAF cells. Additionally, TCZ repressed the ability of CAF cells in promoting epithelial-to-mesenchymal transition, and enhancing the migratory/invasive and proliferative capacities of breast cancer cells in vitro. Importantly, these findings were confirmed in orthotopic humanized breast tumors in mice. Furthermore, TCZ suppressed the expression of the pro-angiogenic factor VEGF-A and its transactivator HIF-1α in CAF cells, and consequently inhibited the angiogenic-promoting effect of active CAFs both in vitro and in orthotopic tumor xenografts. These results indicate that inhibition of the IL-6/STAT3/AUF1 pathway by TCZ can normalize active breast CAFs and suppress their paracrine pro-carcinogenic effects, which paves the way toward development of specific CAF-targeting therapy, badly needed for more efficient breast cancer treatments.

Osteoprotegerin (OPG) mediates the anti-carcinogenic effects of normal breast fibroblasts and targets cancer stem cells through inhibition of the ß-catenin pathway.

Normal breast fibroblasts (NBFs) support and maintain the architecture of the organ, and can also suppress tumorigenesis. However, the mechanisms involved are not fully understood. We have shown here that NBFs suppress breast carcinogenesis through secretion of osteoprotegerin (OPG), a soluble decoy receptor for the Receptor Activator of NF-κB ligand (RANKL). Indeed, NBFs and human recombinant OPG (rOPG), suppressed breast cancer cells proliferation and motility through inhibition of the epithelial-to-mesenchymal transition (EMT) process both in vitro and in vivo. Additionally, rOPG inhibited the IL-6/STAT3 and NF-κB pathways as well as the OPG gene, which turned out to be STAT3-regulated. This was confirmed using denosumab, a RANKL-targeted antibody, which also inhibited NF-κB, down-regulated OPG and repressed EMT in breast cancer cells grown in 2D and 3D. Importantly, both rOPG and denosumab targeted cancer stem cells (CSCs). This was mediated through inhibition of the CSC-related pathway ß-catenin. Moreover, rOPG reduced tumor growth and inhibited breast CSC biomarkers in orthotopic humanized breast tumors. Therefore, normal mammary fibroblasts can suppress carcinogenesis through OPG, which constitutes great potential as preventive and/or therapeutic molecule for breast carcinomas.

AUF1 promotes stemness in human mammary epithelial cells through stabilization of the EMT transcription factors TWIST1 and SNAIL1.

The AU-rich element RNA-binding protein 1 (AUF1) is an RNA-binding protein, which can both stabilize and destabilize the transcripts of several cancer-related genes. Since epithelial-to-mesenchymal transition (EMT) and the acquisition of cancer stem cell traits are important for cancer onset and progression, we sought to determine the role of AUF1 in these two important processes. We have shown that AUF1 induces EMT and stemness in breast epithelial cells via stabilization of the SNAIL1 and TWIST1 mRNAs, and their consequent upregulation. Indeed, AUF1 binds the transcripts of these two genes at their 3'UTR and reduces their turnover. Ectopic expression of AUF1 also promoted stemness in mammary epithelial cells, and thereby increased the proportion of cancer stem cells. Importantly, breast cancer cells that ectopically express AUF1 were more efficient in forming orthotopic tumor xenografts in nude mice than their corresponding controls with limiting cell inocula. On the other hand, AUF1 downregulation with specific siRNA inhibited EMT and reduced the stemness features in breast cancer cells. Moreover, AUF1 knockdown sensitized breast cancer cells to the killing effect of cisplatin. Together, these findings provide clear evidence that AUF1 is an important inducer of the EMT process through stabilization of SNAIL1 and TWIST1 and the consequent promotion of breast cancer stem cells. Thereby, AUF1 targeted molecules could constitute efficient therapeutics for breast cancer patients.

Tocilizumab potentiates cisplatin cytotoxicity and targets cancer stem cells in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a very aggressive subtype with high recurrence rate and no molecular targets for therapies. This subtype is characterized by high expression/secretion of the proinvasive/metastatic interleukin-6 (IL-6) cytokine. In the present study, we have shown that tocilizumab inhibits the IL-6/STAT3 signaling and suppresses the cancer/inflammatory epigenetic IL-6/STAT3/NF-κB positive feedback loop. Furthermore, tocilizumab inhibited the proliferative and the migratory/invasiveness capacities as well as the epithelial-to-mesenchymal transition (EMT) process in TNBC cells. Importantly, tocilizumab suppressed the stemness-related characteristics of TNBC cells, through the inhibition of the Wnt/ß-catenin breast cancer stem cell-related pathway. Additionally, we have shown that tocilizumab suppresses the paracrine activation of normal breast stromal fibroblasts to myofibroblats. Moreover, tocilizumab sensitized TNBC cells to the cytotoxic effect of cisplatin in vitro. Furthermore, pharmacological inhibition of IL-6 by tocilizumab had great inhibitory effect on tumor growth and the EMT process in humanized orthotopic breast tumors in mice. In addition, tocilizumab potentiated the proapoptotic effect of cisplatin in humanized breast tumors. Together, these findings indicate that tocilizumab can suppress the prometastatic capacity of TNBC cells and enhances the cytotoxic effect of cisplatin against these cells. Therefore, tocilizumab could be of great therapeutic value for these hard-to-treat TNBC patients.

The DNA methyl-transferase protein DNMT1 enhances tumor-promoting properties of breast stromal fibroblasts.

The activation of breast stromal fibroblasts is a crucial step toward tumor growth and spread. Therefore, it is extremely important to understand the molecular basis of this activation and determine the molecules and the mechanisms responsible for its sustainability. In the present report we have shown that the DNA methyl-transferase protein DNMT1 is critical for the activation of breast stromal fibroblasts as well as the persistence of their active status. Indeed, we have first revealed DNMT1 up-regulation in most cancer-associated fibroblasts relative to their corresponding adjacent normal fibroblasts. This effect resulted from HuR-dependent stabilization of the DNMT1 mRNA. Furthermore, ectopic expression of DNMT1 activated primary normal breast fibroblasts and promoted their pro-carcinogenic effects, both in vitro and in orthotopic tumor xenografts. By contrast, specific DNMT1 knockdown normalized breast myofibroblasts and repressed their cancer-promoting properties. These effects were sustained through inhibition of the IL-6/STAT3/NF-κB epigenetic cancer/inflammation positive feedback loop. Furthermore, we have shown that DNMT1-related activation of breast fibroblasts is mediated through upregulation of the RNA binding protein AUF1, which is also part of the loop. The present data demonstrate the critical function of DNMT1 in breast cancer-related sustained activation of breast stromal fibroblasts.

Breast stromal fibroblasts from histologically normal surgical margins are pro-carcinogenic.

There is evidence that normal breast stromal fibroblasts (NBFs) suppress tumour growth, while cancer-associated fibroblasts (CAFs) promote tumourigenesis through functional interactions with tumour cells. Little is known about the biology and the carcinogenic potential of stromal fibroblasts present in histologically normal surgical margins (TCFs). Therefore, we first undertook gene expression analysis on five CAF/TCF pairs from breast cancer patients and three NBF samples (derived from mammoplasties). This comparative analysis revealed variation in gene expression between these three categories of cells, with a TCF-specific gene expression profile. This variability was higher in TCFs than in their paired CAFs and also NBFs. Cytokine arrays show that TCFs have a specific secretory cytokine profile. In addition, stromal fibroblasts from surgical margins expressed high levels of α-SMA and SDF-1 and exhibited higher migratory/invasiveness abilities. Indirect co-culture showed that TCF cells enhance the proliferation of non-cancerous mammary epithelial cells and the epithelial-to-mesenchymal transition of breast cancer cells. Moreover, TCF and CAF cells increased the level of PCNA, MMP-2 and the phosphorylated/activated form of Akt in normal breast luminal fibroblasts in a paracrine manner. Furthermore, TCFs were able to promote the formation and growth of humanized orthotopic breast tumours in nude mice. Interestingly, these TCF phenotypes and the extent of their effects were intermediate between those of NBFs and CAFs. Together, these results indicate that stromal fibroblasts located in non-cancerous tissues exhibit a tumour-promoting phenotype, indicating that their presence post-surgery may play important roles in cancer recurrence.

Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers.

BACKGROUND: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts. METHODS: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro. RESULTS: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC. CONCLUSION: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

The nexus between VEGF and NFκB orchestrates a hypoxia-independent neovasculogenesis.

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVß3, α5ß1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.

Inhibition of reactive gliosis prevents neovascular growth in the mouse model of oxygen-induced retinopathy.

Retinal neovascularization (NV) is a major cause of blindness in ischemic retinopathies. Previous investigations have indicated that ischemia upregulates GFAP and PDGF-B expression. GFAP overexpression is a hallmark of reactive gliosis (RG), which is the major pathophysiological feature of retinal damage. In addition, PDGF-B has been implicated in proliferative retinopathies. It was the aim of this study to gain insights on the possible pharmacological interventions to modulate PDGF-B and GFAP expression, and its influence on RG and NV. We used an array of assays to evaluate the effects of YC-1, a small molecule inhibitor of HIF-1 and a novel NO-independent activator of soluble guanylyl cyclase (sGC), on RG and NV, in vivo and in vitro. When compared to the DMSO-treated retinas, dual-intravitreal injections of YC-1, in vivo: (1) suppressed the development and elongation of neovascular sprouts in the retinas of the oxygen-induced retinopathy (OIR) mouse model; and (2) reduced ischemia-induced overexpression of GFAP and PDGF-B at the message (by 64.14±0.5% and 70.27±0.04%) and the protein levels (by 65.52±0.02% and 57.59±0.01%), respectively. In addition, at 100 µM, YC-1 treatment downregulated the hypoxia-induced overexpression of GFAP and PDGF-B at the message level in rMC-1 cells (by 71.42±0.02% and 75±0.03%), and R28 cells (by 58.62±0.02% and 50.00±0.02%), respectively; whereas, the protein levels of GFAP and PDGF-B were reduced (by 78.57±0.02% and 77.55±0.01%) in rMC-1 cells, and (by 81.44±0.02% and 79.16±0.01%) in R28 cells, respectively. We demonstrate that YC-1 reversed RG during ischemic retinopathy via impairing the expression of GFAP and PDGF-B in glial cells. This is the first investigation that delves into the reversal of RG during ischemic retinal vasculopathies. In addition, the study reveals that YC-1 may exert promising therapeutic effects in the treatment of retinal and neuronal pathologies.