scholarly journals Quadruple-editing of MAPK and PI3K pathways effectively blocks the progression of KRAS-mutated colorectal cancer cells

Author(s):  
Zaozao Wang ◽  
Bin Kang ◽  
Qianqian Gao ◽  
Lei Huang ◽  
Yingcong Fan ◽  
...  

Abstract Background Mutated KRAS promotes the activation of mitogen-activated protein kinase (MAPK) pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of phosphatidylinositol 3‑kinase (PI3K) pathway strongly attenuates the efficacy of MAPK suppression in KRAS-mutated CRC cells. The development of a novel strategy targeting dual-pathway is therefore highly essential for the therapy of KRAS-mutated CRC cells. Methods In this study, a quadruple-depleting system for KRAS, MEK1, PIK3CA, and mammalian target of rapamycin (MTOR) genes based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/SaCas9 was developed. Serotype 5 of adenovirus (ADV5) was used as packaging virus for systemic delivery of the CRISPR system. To enhance infection efficiency and specificity of ADV5 to CRC cells and reduce its non-specific tissue tropism, two engineered proteins, an adaptor and a protector were synthesized and formed an ADV-protein complex (APC) when delivered the quadruple-editing system intravenously in vivo. Results The quadruple-editing significantly inhibited MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, the quadruple-editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor protein which specifically targeting epithelial cell adhesion molecule (EpCAM) could dramatically enhance infection efficiency of ADV5 to CRC cells. and the protector protein could significantly reduce the off-targeting tropisms in a variety of organs. Moreover, the quadruple-editing intravenously delivered by APC significantly blocked dual-pathway and tumor growth, without influencing normal tissues in cell- and patient-derived xenograft models of KRAS-mutated CRC cells. Conclusions APC-delivered quadruple-editing of MAPK and PI3K pathways effectively and specifically blocked the progression of KRAS-mutated CRC cells.

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Lorenzo Stramucci ◽  
Angelina Pranteda ◽  
Arianna Stravato ◽  
Carla Azzurra Amoreo ◽  
Annarita Pennetti ◽  
...  

Abstract Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.


2007 ◽  
Vol 176 (5) ◽  
pp. 709-718 ◽  
Author(s):  
Chunxi Ge ◽  
Guozhi Xiao ◽  
Di Jiang ◽  
Renny T. Franceschi

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.


2011 ◽  
Vol 300 (1) ◽  
pp. E103-E110 ◽  
Author(s):  
Xiaoban Xin ◽  
Lijun Zhou ◽  
Caleb M. Reyes ◽  
Feng Liu ◽  
Lily Q. Dong

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C2C12 cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C2C12 myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.


2021 ◽  
Author(s):  
Yunxin Zhang ◽  
Kexin Shen ◽  
Hanyi Zha ◽  
Wentao Zhang ◽  
Haishan Zhang

Abstract BackgroundCircular RNA-BTG3 associated nuclear protein (circ-BANP) was identifified to involve in cell proliferation of colorectal cancer (CRC). The aerobic glycolysis is a key metabolism mediating cancer progression. However, the role of circ-BANP on aerobic glycolysis in CRC remains unknown. MethodsThe expression of circ-BANP, microRNA (miR)-874-3p, and mitogen-activated protein kinase 1 (MAPK1) mNRA was detected using quantitative real-time polymerase chain reaction. Cell viability and invasion were measured by cell counting kit-8 assay or transwell assay. Glucose consumption and lactate production were assessed by a glucose and lactate assay kit. XF Extracellular Flux Analyzer was used to determine extracellular acidifification rate (ECAR). Western blot was used to analyze the levels of hexokinase-2 (HK2), pyruvate kinase M2 (PKM2), MAPK1, proliferating cell nuclear antigen (PCNA), Cyclin D1, N-cadherin, E-cadherin, hypoxia inducible factor-1α (HIF-1α), glucose transport protein 1(GLUT1), and c-Myc. The interaction between miR-874-3p and circ-BANP or MAPK1 was confifirmed by dual luciferase reporter assay. In vivo experiments were conducted through the murine xenograft model. ResultsCirc-BANP was up-regulated in CRC tissues and cell lines. Circ-BANP knockdown suppressed CRC cell proliferation, invasion and aerobic glycolysis in vitro as well as inhibited tumor growth in vivo. Circ-BANP was a sponge of miR-874-3p and performed anti-tumor effffects by binding to miR-874-3p in CRC cells. Subsequently, we confifirmed MAPK1 was a target of miR-874-3p and circ-BANP indirectly regulated MAPK1 expression by sponging miR-874-3p. After that, we found MAPK1 overexpression partially reversed circ-BANP deletion-mediated inhibition on cell carcinogenesis and aerobic glycolysis in CRC. ConclusionCirc-BANP accelerated cell carcinogenesis and aerobic glycolysis by regulating MAPK1 through miR- 874-3p in CRC, suggesting a promising therapeutic strategy for CRC treatment.


1997 ◽  
Vol 17 (3) ◽  
pp. 1702-1713 ◽  
Author(s):  
D D Schlaepfer ◽  
M A Broome ◽  
T Hunter

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.


2021 ◽  
Vol 16 (7) ◽  
pp. 231-239
Author(s):  
Muthu Kumar Thirunavukkarasu ◽  
Ramanathan Karuppasamy

Aberrant stimulation of MAPK (Mitogen-activated protein kinase) signaling pathway triggers the dysregulated cell growth and resistance to apoptosis in a wide variety of tumors especially in NSCLC (Nonsmall cell lung cancer). Most of the research is on treating lung cancer by targeting the MAPK pathway receptors. Nevertheless, it is essential to consider interconnections and mode of action to resolve the drug resistance ad feedback loops during the treatment with checkpoint inhibitors. Here we describe the overall mechanism of MAPK pathway, oncogenic mutations and precise information regarding the drug compounds for each receptor in this pathway. Further, in-depth insights into this review could be beneficial for the empathetic discovery of inhibitors for NSCLC against this pathway.


2017 ◽  
Vol 214 (6) ◽  
pp. 1691-1710 ◽  
Author(s):  
Helen L. Young ◽  
Emily J. Rowling ◽  
Mattia Bugatti ◽  
Emanuele Giurisato ◽  
Nadia Luheshi ◽  
...  

Mitogen-activated protein kinase (MAPK) pathway antagonists induce profound clinical responses in advanced cutaneous melanoma, but complete remissions are frustrated by the development of acquired resistance. Before resistance emerges, adaptive responses establish a mutation-independent drug tolerance. Antagonizing these adaptive responses could improve drug effects, thereby thwarting the emergence of acquired resistance. In this study, we reveal that inflammatory niches consisting of tumor-associated macrophages and fibroblasts contribute to treatment tolerance through a cytokine-signaling network that involves macrophage-derived IL-1β and fibroblast-derived CXCR2 ligands. Fibroblasts require IL-1β to produce CXCR2 ligands, and loss of host IL-1R signaling in vivo reduces melanoma growth. In tumors from patients on treatment, signaling from inflammatory niches is amplified in the presence of MAPK inhibitors. Signaling from inflammatory niches counteracts combined BRAF/MEK (MAPK/extracellular signal–regulated kinase kinase) inhibitor treatment, and consequently, inhibiting IL-1R or CXCR2 signaling in vivo enhanced the efficacy of MAPK inhibitors. We conclude that melanoma inflammatory niches adapt to and confer drug tolerance toward BRAF and MEK inhibitors early during treatment.


2004 ◽  
Vol 24 (3) ◽  
pp. 1081-1095 ◽  
Author(s):  
Nicole H. Purcell ◽  
Dina Darwis ◽  
Orlando F. Bueno ◽  
Judith M. Müller ◽  
Roland Schüle ◽  
...  

ABSTRACT The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.


2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.


2008 ◽  
Vol 412 (3) ◽  
pp. 435-445 ◽  
Author(s):  
Maja Jensen ◽  
Jane Palsgaard ◽  
Rehannah Borup ◽  
Pierre de Meyts ◽  
Lauge Schäffer

Single-chain peptides have been recently produced that display either mimetic or antagonistic properties against the insulin and IGF-1 (insulin-like growth factor 1) receptors. We have shown previously that the insulin mimetic peptide S597 leads to significant differences in receptor activation and initiation of downstream signalling cascades despite similar binding affinity and in vivo hypoglycaemic potency. It is still unclear how two ligands can initiate different signalling responses through the IR (insulin receptor). To investigate further how the activation of the IR by insulin and S597 differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK (mitogen-activated protein kinase) pathway. These findings support our signalling results obtained previously and confirm that the main difference between S597 and insulin stimulation resides in the activation of the MAPK pathway. In conclusion, we show that insulin and S597 acting via the same receptor differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications.


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