scholarly journals Sirolimus plus nintedanib treats vascular pathology in HHT mouse models

2019 ◽  
Author(s):  
Santiago Ruiz ◽  
Haitian Zhao ◽  
Pallavi Chandakkar ◽  
Julien Papoin ◽  
Hyunwoo Choi ◽  
...  
Keyword(s):  
Receptor Tyrosine Kinase ◽  
Arteriovenous Malformations ◽  
Mtor Inhibitor ◽  
Kinase Inhibitor ◽  
Therapeutic Benefit ◽  
Vascular Pathology ◽  
Loss Of Function ◽  
Adult Mice ◽  
Retinal Bleeding

ABSTRACTHereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1-ENG-Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K-Akt-mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine-kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs—including in HHT patient blood outgrowth ECs—and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in HHT patients.

scholarly journals CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jin-Chun Qi ◽  
Zhan Yang ◽  
Tao Lin ◽  
Long Ma ◽  
Ya-Xuan Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activation ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Biological Effects ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Positive Feedback Loop

Abstract Background Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa. Methods The expression levels of CDK13 in PCa tissues and different cell lines were determined by quantitative real-time PCR and Western blot analysis. In vitro and in vivo assays were preformed to explore the biological effects of CDK13 in PCa cells. Co-immunoprecipitation anlysis coupled with mass spectrometry was used to identify E2F5 interaction with CDK13. A CRISPR-Cas9 complex was used to activate endogenous CDK13 and circCDK13 expression. Furthermore, the mechanism of circCDK13 was investigated by using loss-of-function and gain-of-function assays in vitro and in vivo. Results Here we show that CDK13 is significantly upregulated in human PCa tissues. CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5. Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation. Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation. Conclusions These findings suggest that CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 is responsible for PCa development. Targeting this newly identified regulatory axis may provide therapeutic benefit against PCa progression and drug resistance.

scholarly journals Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration

2017 ◽  
Vol 114 (10) ◽  
pp. E2006-E2015 ◽  
Author(s):  
János Hanics ◽  
Edit Szodorai ◽  
Giuseppe Tortoriello ◽  
Katarzyna Malenczyk ◽  
Erik Keimpema ◽  
...  
Keyword(s):  
Annexin V ◽  
Therapeutic Benefit ◽  
Mouse Genetics ◽  
Matrix Metalloprotease ◽  
Loss Of Function ◽  
Neuroblast Migration ◽  
Sensor Protein ◽  
Migratory Stream

The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

scholarly journals A novel treatment strategy for lapatinib resistance in a subset of HER2-amplified gastric cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Gang Ning ◽  
Qihui Zhu ◽  
Wonyoung Kang ◽  
Hamin Lee ◽  
Leigh Maher ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Pharmacological Study ◽  
Mapk Signaling ◽  
Her2 Overexpression ◽  
Loss Of Function

Abstract Background Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 13–23% of all GC cases and patients with HER2 overexpression exhibit a poor prognosis. Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, is an effective agent to treat HER2-amplified breast cancer but it failed in gastric cancer (GC) clinical trials. However, the molecular mechanism of lapatinib resistance in HER2-amplified GC is not well studied. Methods We employed an unbiased, genome-scale screening with pooled CRISPR library on HER2-amplified GC cell lines to identify genes that are associated with resistance to lapatinib. To validate the candidate genes, we applied in vitro and in vivo pharmacological tests to confirm the function of the target genes. Results We found that loss of function of CSK or PTEN conferred lapatinib resistance in HER2-amplified GC cell lines NCI-N87 and OE19, respectively. Moreover, PI3K and MAPK signaling was significantly increased in CSK or PTEN null cells. Furthermore, in vitro and in vivo pharmacological study has shown that lapatinib resistance by the loss of function of CSK or PTEN, could be overcome by lapatinib combined with the PI3K inhibitor copanlisib and MEK inhibitor trametinib. Conclusions Our study suggests that loss-of-function mutations of CSK and PTEN cause lapatinib resistance by re-activating MAPK and PI3K pathways, and further proved these two pathways are druggable targets. Inhibiting the two pathways synergistically are effective to overcome lapatinib resistance in HER2-amplified GC. This study provides insights for understanding the resistant mechanism of HER2 targeted therapy and novel strategies that may ultimately overcome resistance or limited efficacy of lapatinib treatment for subset of HER2 amplified GC.

Inhibiting the IGF-1 Receptor Tyrosine Kinase with Picropodophillin: An In Vitro and In Vivo Study in the 5T33MM Mouse Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 640-640
Author(s):  
Karin Vanderkerken ◽  
Eline Menu ◽  
Thomas Stromberg ◽  
Hendrik De Raeve ◽  
Kewal Asosingh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Microvessel Density ◽  
Kinase Inhibitor ◽  
High Dose

Abstract Multiple myeloma (MM) represents a B-cell malignancy, characterized by monoclonal proliferation of plasma cells in the bone marrow (BM) and is associated with osteolysis and angiogenesis. Insulin-like growth factor-1 (IGF-1), produced by the BM stromal cells, has been described as an important factor in the survival, proliferation and migration of MM cells. The latter process is involved in the homing of the MM cells to the BM. IGF-1 also induces VEGF secretion by the MM cells, thus stimulating angiogenesis in the BM. As IGF-1 is a pleiotropic factor in MM, therapeutic strategies targeting the IGF-1R may be effective as anti-tumor treatments. In this work we investigated the effect of an IGF-1 receptor tyrosine kinase inhibitor (picropodophyllin or PPP1) in the murine, syngeneic 5T33MM model of multiple myeloma. This mouse model is representative for the human disease and can combine in vitro and in vivo studies. We first investigated the effects of PPP on the MM cells in vitro. We and others have previously demonstrated that IGF-1 induced ERK activation, involved in VEGF secretion and proliferation. When the 5T33MM cells were preincubated with 1microM PPP, Western blot analysis demonstrated the blocking of this activation. Furthermore, when the 5T33MM cells were preincubated with PPP for 30 min, IGF-1 induced VEGF secretion and proliferation of the 5T33MM cells were completely blocked. Next, we used the tyrosine kinase inhibitor PPP in vivo. 5T33MM cells were injected intravenously in C57BLKaLwRij mice and the development of the disease was monitored by measuring the serum paraprotein concentration. Mice were either treated with a low (17mM, IP, twice a day) or a high dose of PPP (50mM, IP, twice a day) or with the vehicle (DMSO/oil 9/1) from the day of injection with 5T33MM onward. At week 3, vehicle controls showed signs of morbidity and were sacrificed. The presence of tumor was measured by assessing serum paraprotein concentrations and determining the proportion of idiotype positive cells in the BM by flow cytometry. Angiogenesis was assessed by measuring the microvessel density on CD31 stained paraffin sections. The tumor burden in the bone marrow in the PPP treated mice was 77% lower than in vehicle treated animals (p< 0,0001) and the serum paraprotein concentration was 90% lower (p< 0,0001). The microvessel density in the BM of the PPP treated group was reduced by 60% (p< 0,02). In a separate survival experiment the mice were either treated with the vehicle or with the high dose (50mM) of PPP, from the time of tumor injection. Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with PPP when compared with vehicle (28 vs. 18 days, p<0,001). These data demonstrate that the IGF-1RTK inhibitor PPP possesses strong anti-tumor activity, as demonstrated both in vitro and in vivo in a syngeneic model of multiple myeloma, and may therefore be an effective therapeutic candidate for MM treatment.

Multi-Targeted Receptor Tyrosine Kinase Inhibitor, ABT-869, Induces Apoptosis and Suppresses Proliferation of Ba/F3 FLT-3 ITD Mutant Cells in Vitro and in Vivo through Inhibition of FLT3 and AKT.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1938-1938
Author(s):  
Jenny E Hernandez ◽  
Joan Zape ◽  
Keith Glaser ◽  
Elliot Landaw ◽  
Cecilia Fu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Vehicle Control ◽  
Ic50 Value ◽  
Mutant Cells

Abstract FLT3 is a receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with acute myelogenous leukemia (AML) and confer a poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations in the FLT3 kinase activation loop, e.g. D835V. We are studying the signaling pathways associated with a small molecule multi-targeted receptor tyrosine kinase inhibitor (RTKI), ABT-869. To determine the effects of ABT-869 in vitro and in vivo, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation was used as an isolated FLT-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells (IC50 value of 1 nM) when compared to Ba/F3 Flt-3 D835V mutant (IC50 value between 1 and 10 μM) and Ba/F3 Flt-3 wildtype (WT) cells (IC50 value of 10 μM). Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis occurred in Ba/F3 Flt-3 ITD mutant cells treated with 1μM ABT-869 for 24 hours (42.8%) when compared to untreated (4.7%) or vehicle control (4.0%) cells. Ba/F3 Flt-3 D835V mutant cell lines demonstrated a 12.5% rate of apoptosis at 1μM, compared to untreated (1.99%) and vehicle control (2.1%) cell lines. Propidium iodide staining of treated Ba/F3 Flt-3 WT cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cells or DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells, following 6 hours of treatment with 1 to 100 nM ABT-869, whereas no cleavage was observed in Ba/F3 WT cells treated with ABT-869. To study the effects of ABT-869 in vivo, we treated SCID mice injected with Ba/F3 Flt-3 ITD, Ba/F3 Flt-3 D835V, or Ba/F3 Flt-3 WT cells and monitored disease progression using bioluminescence imaging. The mice injected with the Ba/F3 FLT-3 ITD mutant cells and treated with vehicle control developed metastases and had a median survival time of 2 weeks. In contrast, the ABT-869 treated group had slower disease progression with median survival of 6.2 weeks (P&lt;0.008). Both control and treated mice injected with Ba/F3 FLT-3 D835V mutant cell lines developed metastases and had similar survival (median 1.7 and 1.9 weeks, respectively). Survival times of control and treated mice injected with Ba/F3 FLT-3 WT cells were also similar (median 8.4 and 8.1 weeks, respectively). Previous work identified that ABT-869 induced apoptosis of acute myeloid leukemia cells through inhibition of FLT-3 reception phosphorylation, which is observed as early as 3 hours after treatment. In Ba/F3 cells expressing FLT-3 ITD, ABT-869 also inhibited phosphorylation of AKT, which is upstream of the pro-apoptotic protein Bad. Therefore, our preclinical data suggest that ABT-869 induces apoptosis of FLT-3 ITD mutant cells both in vitro and in vivo. These studies provide rationale for the treatment of acute leukemia patients harboring the FLT3-ITD mutation with ABT-869 and the potential benefit of combining small molecule inhibitors that target both RTKs and AKT.

Sign in / Sign up

Export Citation Format

Share Document