Hypoxic TAM-derived exosomal miR-155-5p promotes RCC progression through HuR-dependent IGF1R/AKT/PI3K pathway

AbstractHypoxic tumor-associated macrophages (TAMs) are related to poor prognosis of patients with clear cell renal cell carcinoma (ccRCC). Exosomes are small lipid-bilayer vesicles that implicated in tumor progression and metastasis. However, whether hypoxic TAM-derived exosomes affect RCC progression within the hypoxic tumor microenvironment has not been elucidated. GSE analysis identified miR-155-5p was upregulated in RCC. Moreover, we quantified levels of miR-155-5p using RT-qPCR, performed immunohistochemical staining in 79 pairs of primary RCC specimens and related them to clinicopathological parameters. Higher miR-155-5p levels were related to more CD163 + TAM infiltration and elevated HIF-1a expression in our cohort. In the in vitro studies, we initially purified and characterized the exosomes from the supernatant of TAMs subjected to normoxia or hypoxia, and then transfected antagomiR-155-5p or control into these TAMs to produce corresponding exosomes. Gain and loss-of-function studies further investigated the effect of transferred hypoxic exosomal miR-155-5p on the cross-talk between TAMs and RCC cells in xenograft model and in vitro co-culture experiments. The results of RNA immunoprecipitation analyses elucidated that miR-155-5p could directly interact with human antigen R (HuR), thus increasing IGF1R mRNA stability. Mechanistically, hypoxic TAM-Exo transferred miR-155-5p promoted RCC progression partially through activating IGF1R/PI3K/AKT cascades. Taken together, transfer of miR-155-5p from hypoxic TAMs by exosomes to renal cancer cells explains the oncogenic manner, in which M2 macrophages confer the malignant phenotype to RCC cells by enhancing HuR-mediated mRNA stability of IGF1R.

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Mir-144-3p Promotes Cell Proliferation, Metastasis, Sunitinib Resistance in Clear Cell Renal Cell Carcinoma by Downregulating ARID1A

Cellular Physiology and Biochemistry ◽

10.1159/000484395 ◽

2017 ◽

Vol 43 (6) ◽

pp. 2420-2433 ◽

Cited By ~ 35

Author(s):

Wen Xiao ◽

Ning Lou ◽

Hailong Ruan ◽

Lin Bao ◽

Zhiyong Xiong ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Proliferation ◽

Cell Carcinoma ◽

Renal Cell ◽

Clear Cell ◽

Xenograft Model ◽

Luciferase Reporter ◽

Loss Of Function ◽

Cell Renal Cell Carcinoma

Background/Aims: We previously performed microRNA (miRNA) microarray to identify effective indicators of clear cell renal cell carcinoma (ccRCC) tissue samples and preoperative/postoperative plasma in which we identified miR-144-3p as an oncomiRNA. However, the molecular mechanism of miR-144-3p remains unclear. This study aims to explore the roles of miR-144-3p in the invasion, migration and Sunitinib-resistance in ccRCC and to elucidate the underlying mechanisms. Methods: Gain and loss of function approaches were used to investigate the cell proliferation, cycle distribution, clonogenicity, migration, invasion, chemosensitivity of miR-144-3p in vitro. The xenograft model was used to assess the effects of miR-144-3p overexpression on tumorigenesis. Bioinformatics analysis and dual-luciferase reporter assay were used to indentify AT-rich interactive domain 1A (ARID1A) as a direct target gene of miR-144-3p. Quantitative RT-PCR, Western blotting, and immunohistochemical (IHC) staining were used to explore ARID1A expression level of the mRNA and protein. Results: We found that miR-144-3p overexpression enhanced cell proliferation, clonogenicity, migration, invasion, and chemoresistance in ccRCC cells. Notably, the oncotumor activities of miR-144-3p were mediated by repressing the expression of ARID1A. The downregulation of ARIDIA could promote the function of miR-144-3p in cell proliferation, metastasis and chemoresistance. Consistently, ARID1A mRNA and protein levels were decreased in ccRCC and in nude mice, and they negatively correlated with miR-144-3p. Conclusion: Higher miR-144-3p may enhance malignancy and resistance to Sunitinib in ccRCC by targeting ARID1A, the observations may uncover novel strategies of ccRCC treatment.

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Warburg effect-promoted exosomal circ_0072083 releasing up-regulates NANGO expression through multiple pathways and enhances temozolomide resistance in glioma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01942-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Chenyu Ding ◽

Xuehan Yi ◽

Xiangrong Chen ◽

Zanyi Wu ◽

Honghai You ◽

...

Keyword(s):

Warburg Effect ◽

Lactate Production ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Rna Immunoprecipitation ◽

Resistant Cells

Abstract Background Temozolomide (TMZ) resistance limits its application in glioma. Exosome can carry circular RNAs (circRNAs) to regulate drug resistance via sponging microRNAs (miRNAs). miRNAs can control mRNA expression by regulate the interaction with 3’UTR and methylation. Nanog homeobox (NANOG) is an important biomarker for TMZ resistance. Hitherto, it is unknown about the role of exosomal hsa_circ_0072083 (circ_0072083) in TMZ resistance in glioma, and whether it is associated with NANOG via regulating miRNA sponge and methylation. Methods TMZ-resistant (n = 36) and sensitive (n = 33) patients were recruited. The sensitive cells and constructed resistant cells were cultured and exposed to TMZ. circ_0072083, miR-1252-5p, AlkB homolog H5 (ALKBH5) and NANOG levels were examined via quantitative reverse transcription polymerase chain reaction and western blot. The half maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, migration and invasion were analyzed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing and transwell assays. The in vivo function was assessed using xenograft model. The N6-methyladenosine (m6A) level was analyzed via methylated RNA immunoprecipitation (MeRIP). Target relationship was investigated via dual-luciferase reporter assay and RNA immunoprecipitation. Warburg effect was investigated via lactate production, glucose uptake and key enzymes expression. Exosome was isolated and confirmed via transmission electron microscopy and specific protein expression. Results circ_0072083 expression was increased in TMZ-resistant glioma tissues and cells. circ_0072083 knockdown restrained the resistance of resistant cells via decreasing IC50 of TMZ, proliferation, migration, invasion and xenograft tumor growth and increasing apoptosis. circ_0072083 silence reduced NANOG expression via blocking ALKBH5-mediated demethylation. circ_0072083 could regulate NANOG and ALKBH5 via targeting miR-1252-5p to control TMZ resistance. Warburg effect promoted the release of exosomal circ_0072083 in resistant cells. Exosomal circ_0072083 from resistant cells increased the resistance of sensitive cells to TMZ in vitro and xenograft model. Exosomal circ_0072083 level was enhanced in resistant patients, and it had a diagnostic value and indicated a lower overall survival in glioma. Conclusion Exosomal circ_0072083 promoted TMZ resistance via increasing NANOG via regulating miR-1252-5p-mediated degradation and demethylation in glioma.

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LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01854-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Gege Shu ◽

Huizhao Su ◽

Zhiqian Wang ◽

Shihui Lai ◽

Yan Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Xenograft Model ◽

Luciferase Reporter ◽

Loss Of Function ◽

Mechanism Study ◽

Downstream Signaling ◽

Mouse Xenograft Model ◽

High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

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TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro

BioMed Research International ◽

10.1155/2020/8409239 ◽

2020 ◽

Vol 2020 ◽

pp. 1-18

Author(s):

Yingkun Xu ◽

Guangzhen Wu ◽

Jiayao Zhang ◽

Jianyi Li ◽

Ningke Ruan ◽

...

Keyword(s):

Tumor Growth ◽

Mrna Expression ◽

Wnt Signaling Pathway ◽

Xenograft Model ◽

The Cancer Genome Atlas ◽

Migration And Invasion ◽

Cell Renal Cell Carcinoma ◽

Expression Levels

Purpose. To evaluate the expression of tripartite motif-containing 33 (TRIM33) in ccRCC tissues and explore the biological effect of TRIM33 on the progress of ccRCC. Method. The Cancer Genome Atlas (TCGA) database was used to examine the mRNA expression levels of TRIM33 in ccRCC tissues and its clinical relevance. Immunohistochemistry (IHC) was performed to evaluate its expression in ccRCC tissues obtained from our hospital. The correlation between TRIM33 expression and clinicopathological features of the patients was also investigated. The effects of TRIM33 on the proliferation of ccRCC cells were examined using the CCK-8 and colony formation assays. The effects of TRIM33 on the migration and invasion of ccRCC cells were explored through wound healing and transwell assays, along with the use of Wnt signaling pathway agonists in rescue experiments. Western blotting was used to explore the potential mechanism of TRIM33 in renal cancer cells. A xenograft model was used to explore the effect of TRIM33 on tumor growth. Result. Bioinformatics analysis showed that TRIM33 mRNA expression in ccRCC tissues was downregulated, and low TRIM33 expression was related to poor prognosis in ccRCC patients. In agreement with this, low TRIM33 expression was detected in human ccRCC tissues. TRIM33 expression levels were correlated with clinical characteristics, including tumor size and Furman’s grade. Furthermore, TRIM33 overexpression inhibited proliferation, migration, and invasion of 786-O and ACHN cell lines. The rescue experiment showed that the originally inhibited migration and invasion capabilities were restored. TRIM33 overexpression reduced the expression levels of β-catenin, cyclin D1, and c-myc, and inhibited tumor growth in ccRCC cells in vivo. Conclusion. TRIM33 exhibits an abnormally low expression in human ccRCC tissues. TRIM33 may serve as a potential therapeutic target and prognostic marker for ccRCC.

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LncRNA LINC00342 contributes to the growth and metastasis of colorectal cancer via targeting miR-19a-3p/NPEPL1

10.21203/rs.3.rs-35112/v1 ◽

2020 ◽

Author(s):

Peng Shen ◽

Lili Qu ◽

Jingjing Wang ◽

Quchen Ding ◽

Chuanwen Zhou ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Xenograft Model ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Protein Coding ◽

Mechanistic Investigation ◽

Rna Immunoprecipitation

Abstract Background Long intergenic non-protein coding RNA 342 (LINC00342) has been identified as a novel oncogene, however, the functional role of LINC00342 in colorectal cancer (CRC) remained unclear. Methods The expression of LINC00342 was detected by real-time PCR. Cell proliferation, migration and invasion and xenograft model were examined to analyze the biological functions of LINC00342 in vitro and in vivo. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to identify the target interactions between LINC00342, miR-19a-3p and aminopeptidase like 1 (NPEPL1). Results LINC00342 was highly expressed in CRC. Downregulation of LINC00342 inhibited cell proliferation and metastasis of CRC cells. Moreover, knocking down LINC00342 could weaken the tumor growth in vivo. Mechanistic investigation revealed that LINC00342 may sponge miR-19a-3p to regulate NPEPL1 expression. Further investigation indicated that the oncogenesis facilitated by LINC00342 was inhibited by NPEPL1 depletion.Conclusion LINC00342 promoted CRC progression by competitively binding miR-19a-3p with NPEPL1.

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Linc00473 potentiates cholangiocarcinoma progression by modulation of DDX5 expression via miR-506 regulation

10.21203/rs.3.rs-25881/v2 ◽

2020 ◽

Author(s):

Lining Huang ◽

Xingming Jiang ◽

Zhenglong Li ◽

Jinglin Li ◽

Xuan Lin ◽

...

Keyword(s):

Cell Lines ◽

Cancer Progression ◽

Luciferase Reporter ◽

Loss Of Function ◽

Qrt Pcr ◽

Competitive Endogenous Rnas ◽

Rna Immunoprecipitation ◽

Underlying Mechanisms

Abstract Background: Cholangiocarcinoma (CCA) is a mortal cancer with high mortality, whereas the function and mechanism of occurrence and progression of CCA are still mysterious. Long non-coding RNAs (lncRNAs) could function as important regulators in carcinogenesis and cancer progression. Growing evidences have indicated that the novel lncRNA linc00473 plays an important role in cancer progression and metastasis. However, its function and molecular mechanism in CCA remain unknown. Methods: The linc00473 expression in CCA tissues and cell lines was analyzed using qRT-PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of linc00473 both in vitro and in vivo. Insights into the underlying mechanisms of competitive endogenous RNAs (ceRNAs) were determined by bioinformatics analysis, dual-luciferase reporter assays, qRT-PCR arrays, RNA immunoprecipitation (RIP) and rescue experiments. Results: Linc00473 was highly expressed in CCA tissues and cell lines. Linc00473 knockdown inhibited CCA growth and metastasis. Furthermore, linc00473 acted as miR-506 sponge and regulated its target gene DDX5 expression. Rescue assays verified that linc00473 modulated the tumorigenesis of CCA by regulating miR-506. Conclusions: The data indicated that linc00473 played an oncogenic role in CCA growth and metastasis, and could serve as a novel molecular target for treating CCA.

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Long Non-Coding RNA NEAT1 Promotes HCC Progression via PKM2 and Exosome-Mediated Transfer

10.21203/rs.3.rs-1038606/v1 ◽

2021 ◽

Author(s):

Junping Pan ◽

Yingzhe Hu ◽

Chenlu Yuan ◽

Yafu Wu ◽

Xinhua Zhu

Keyword(s):

Malignant Tumors ◽

Xenograft Model ◽

Migration And Invasion ◽

Mouse Xenograft Model ◽

Rna Immunoprecipitation ◽

Tumor Growth And Metastasis ◽

Lncrna Neat1 ◽

Hcc Cells

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high mortality and poor prognosis. Long non-coding RNAs NEAT1 (lncRNA NEAT1) have been found to play an important role in HCC progression. However, the role and potential molecular mechanism of lncRNA NEAT1 in HCC remain largely unclear. Methods The role of lncRNA NEAT1 both in vitro and in vivo was investigated, with RNA pull-down and RNA immunoprecipitation (RIP) assays being performed to determine the interaction among NEAT1 and FOXO3 and PKM2. In addition, HCC cells were treated with exosomes derived from NEAT1-overexpressing HCC cells, and then cell proliferation, migration and invasion were assessed using in vitro assays. Results In this study, overexpression of NEAT1 promoted the proliferation, migration and invasion of HCC cells, whereas NEAT1 knockdown exhibited the opposite effects. Mechanistically, NEAT1 was found to recruit transcription factor FOXO3 to PKM2 promoter region and upregulate PKM2 expression. Meanwhile, overexpression of NEAT1 increased tumor growth and metastasis in a mouse xenograft model of HCC in vivo via upregulation of PKM2. Furthermore, overexpression of NEAT1 promoted exosome release from HCC cells. Exosomes secreted from NEAT1-overexpressing HCC cells promoted the proliferation, migration and invasion of HCC cells. Conclusion We found that NEAT1 could promote HCC progression via upregulation of PKM2 and exosome-mediated transfer. These data indicated that NEAT1 may be a therapeutic target in HCC.

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VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma

Science ◽

10.1126/science.aap8411 ◽

2018 ◽

Vol 361 (6399) ◽

pp. 290-295 ◽

Cited By ~ 32

Author(s):

Jing Zhang ◽

Tao Wu ◽

Jeremy Simon ◽

Mamoru Takada ◽

Ryoichi Saito ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Clear Cell ◽

Nuclear Factor Κb ◽

Loss Of Function ◽

Cell Renal Cell Carcinoma ◽

A Genome

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

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The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

Journal of the American Society of Nephrology ◽

10.1681/asn.2017121338 ◽

2018 ◽

Vol 29 (8) ◽

pp. 2110-2122 ◽

Cited By ~ 14

Author(s):

Gentzon Hall ◽

Brandon M. Lane ◽

Kamal Khan ◽

Igor Pediaditakis ◽

Jianqiu Xiao ◽

...

Keyword(s):

Biochemical Characterization ◽

Adaptor Protein ◽

Pi3k Pathway ◽

Actin Binding ◽

Loss Of Function ◽

Signaling Axis ◽

Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

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Disruption of Src function potentiates Chk1-inhibitor–induced apoptosis in human multiple myeloma cells in vitro and in vivo

Blood ◽

10.1182/blood-2010-06-291146 ◽

2011 ◽

Vol 117 (6) ◽

pp. 1947-1957 ◽

Cited By ~ 23

Author(s):

Yun Dai ◽

Shuang Chen ◽

Rena Shah ◽

Xin-Yan Pei ◽

Li Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Xenograft Model ◽

Mitogen Activated Protein Kinase ◽

Loss Of Function ◽

Compensatory Response ◽

Human Multiple Myeloma ◽

Chk1 Inhibitor ◽

Induced Apoptosis

Abstract Ras/MEK/ERK pathway activation represents an important compensatory response of human multiple myeloma (MM) cells to checkpoint kinase 1 (Chk1) inhibitors. To investigate the functional roles of Src in this event and potential therapeutic significance, interactions between Src and Chk1 inhibitors (eg, UCN-01 or Chk1i) were examined in vitro and in vivo. The dual Src/Abl inhibitors BMS354825 and SKI-606 blocked Chk1-inhibitor–induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, markedly increasing apoptosis in association with BimEL up-regulation, p34cdc2 activation, and DNA damage in MM cell lines and primary CD138+ MM samples. Loss-of-function Src mutants (K297R, K296R/Y528F) or shRNA knock-down of Src prevented the ERK1/2 activation induced by Chk1 inhibitors and increased apoptosis. Conversely, constitutively active Ras or mitogen-activated protein kinase/ERK kinase 1 (MEK1) significantly diminished the ability of Src inhibitors to potentiate Chk1-inhibitor lethality. Moreover, Src/Chk1-inhibitor cotreatment attenuated MM-cell production of vascular endothelial growth factor and other angiogenic factors (eg, ANG [angiogenin], TIMP1/2 [tissue inhibitor of metalloproteinases 1/2], and RANTES [regulated on activation normal T-cell expressed and secreted]), and inhibited in vitro angiogenesis. Finally, coadministration of BMS354825 and UCN-01 suppressed human MM tumor growth in a murine xenograft model, increased apoptosis, and diminished angiogenesis. These findings suggest that Src kinase is required for Chk1-inhibitor–mediated Ras → ERK1/2 signaling activation, and that disruption of this event sharply potentiates the anti-MM activity of Chk1 inhi-bitors in vitro and in vivo.

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