Activation Of MAPK Pathway Research Articles

466P Efficacy of BRAF inhibitor FORE8394 in BRAF V600+ patients

FORE8394 is a selective, potent BRAFi (class 1, 2 including fusions). By disrupting BRAF dimers, FORE8394 evades paradoxical MAPK pathway activation and may be less prone to toxicities & resistance typical of 1st generation BRAFi.

GSTM2 is a key molecular determinant of resistance to SG-ARIs.

Prostate cancer (PCa) continues to threaten men's health, and treatment targeting the androgen receptor (AR) pathway is the major therapy for PCa patients. Several second-generation androgen receptor inhibitors (SG-ARIs), including enzalutamide (ENZ), apalutamide (APA) and darolutamide (DARO), have been developed to better block the activity of AR. Unavoidably, emergence of resistance to these novel drugs still persists. Herein, we identified glutathione S-transferase Mu 2 (GSTM2) as an important determinant in the acquisition of resistance to SG-ARIs. Elevated GSTM2 was detected in enzalutamide-resistant (ENZ-R) PCa, and overexpression of GSTM2 in naïve enzalutamide-sensitive (ENZ-S) cells effectively transformed them to ENZ-R PCa. Aryl hydrocarbon receptor (AhR), the upstream transcription factor, was implicated in the overexpression of GSTM2 in ENZ-R cells. Mechanistically, GSTM2 antagonized the effect of ENZ by rescuing cells from oxidative stress-associated damage and activation of p38 MAPK pathway. Surprisingly, high GSTM2 levels also associated with cross-resistance to APA and DARO. Taking together, these results provide new insight to ameliorate resistance to SG-ARIs and improve treatment outcome.

Arsenic compounds activate MAPK and inhibit Akt pathways to induce apoptosis in MA-10 mouse Leydig tumor cells.

Arsenic compounds have been applied treating acute promyelocytic 1eukemia and solid tumors with brief mechanism investigations. In fact, we have demonstrated that sodium arsenite plus dimethylarsenic acid could activate apoptosis in MA-10 mouse Leydig tumor cells by inducing caspase pathways. However, detail underlying mechanisms how caspase cascade is regulated remains elusive. Therefore, the apoptotic mechanism of sodium arsenite plus dimethylarsenic acid were examined in MA-10 cells in this study. Our results reveal that Fas/FasL protein expressions were stimulated by sodium arsenite plus dimethylarsenic acid in MA-10 cells. In addition, reactive oxygen species (ROS) generation, cytochrome C release, Bid truncation, and Bax translocation were induced in MA-10 cells by arsenic compounds. Moreover, activation of p38, JNK and ERK1/2, MAPK pathways was stimulated while Akt phosphorylated levels and Akt expression were decreased by sodium arsenite plus dimethylarsenic in MA-10 cells. In conclusion, sodium arsenite and dimethylarsenic acid did activate MAPK pathway plus ROS generation, but suppress Akt pathway, to modulate caspase pathway and then induce MA-10 cell apoptosis.

MEK inhibitor sensitivity in BRAF fusion-driven prostate cancer.

The identification of subpopulations harboring druggable targets has become a major step forward in the subclassification of solid tumors into small groups suitable for specific therapies. BRAF fusions represent a paradigm of uncommon and targetable oncogenic events and have been widely correlated to the development of specific malignancies. However, they are only present in a limited frequency across most common tumor types. At this regard, we performed a genomic screening aimed to identifying rare variants associated to advanced prostate cancer development. Tumoral tissue genomic screening of 41 patients developing advanced prostate cancer was performed at our center as part of the GETHI XX study. The project, sponsored by the Spanish Collaborative Group in Rare Cancers (GETHI), aims to analyze the molecular background of rare tumors and to discover unfrequent molecular variants in common tumors. Here we present the clinical outcome and an in-deep molecular analysis performed in a case harboring a SND1-BRAF fusion gene. The identification of such rearrangement in a patient refractory to standard therapies led to the administration of trametinib (MEK inhibitor). Despite unsensitive to standard therapies, the patient achieved a dramatic response to trametinib. A comprehensive study of the tumor demonstrated this event to be a trunk alteration with higher expression of MEK in areas of tumor invasion. Our study describes the patient-driven discovery of the first BRAF fusion-driven prostate cancer effectively treated with trametinib. Consequently, MAPK pathway activation could define a new subtype of prostate cancer susceptible to a tailored management.

CPNE1 silencing inhibits cell proliferation and accelerates apoptosis in human gastric cancer

Gastric cancer is a heterogeneous disease accompanied by the alteration of various causative genes. The discovery of molecular targets and potential mechanisms of gastric cancer is valuable. Here we explored the biological function of CPNE1 and its molecular mechanisms in gastric cancer. Immunohistochemistry and Kaplan–Meier plotter database were used to identify that CPNE1 was upregulated in human gastric cancer and high expression of CPNE1 suggested a worse prognosis. Silencing CPNE1 could effectively suppress tumor proliferation, accelerate cell apoptosis and arrest cell cycle in vitro. CPNE1 knockdown mediating apoptosis by PARP-1 cleavage via caspase-3 and -7 activation through cytochrome c release from mitochondria in gastric cancer cells. Xenograft mouse model showed that targeted inhibition of CPNE1 slowed down the rate of tumor growth in vivo. We also verified that CPNE1 knockdown inhibited the activation of MAPK pathway mediated by DDIT3-FOS-MKNK2 axis. Specific inhibitor of DDIT3-FOS-MKNK2 axis could suppress gastric cancer cell proliferation, concomitant with knockdown of CPNE1. In conclusion, CPNE1 silencing inhibited gastric cancer growth via deactivating DDIT3-FOS-MKNK2 axis, which indicated that CPNE1 might serve as a therapeutic target for gastric cancer.

BMAL1 promotes colorectal cancer cell migration and invasion through ERK- and JNK-dependent c-Myc expression.

Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT-like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial-to-mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK-8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial-to-mesenchymal transition (EMT) pathway and induce the β-catenin release further promotes the expression of oncogene c-Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK-signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c-Myc in vitro. Taken together, these results suggested that the BMAL1/ c-Myc-signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK-dependent pathway. Our study showed that BMAL1 promotes CRC metastasis through MAPK-c-Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

Salidroside alleviates severe acute pancreatitis-triggered pancreatic injury and inflammation by regulating miR-217-5p/YAF2 axis

BackgroundOur previous studies have shown that salidroside (Sal) exerted a protective effect in severe acute pancreatitis (SAP) via inhibiting the inflammatory response. However, the molecular mechanism has not been fully elucidated. MethodsUsing SAP rat model and miRNA microarray, the effect of Sal on miRNA expression profiling was determined and then validated their changes by quantitative Real-time PCR (qRT-PCR). Then, SAP cell model, enzyme-linked immunosorbent assay (ELISA) and Cell Counting Kit-8 (CCK-8) assay were used to explore the biological function of miR-217-5p in vitro. Bioinformatics analysis, luciferase reporter assay and miRNA pulldown assay were performed to investigate the underlying mechanism of miR-217-5p in the protection of Sal against SAP. ResultsCompared with SAP group, 21 differentially expressed miRNAs were identified in SAP + Sal group. The target genes of these miRNAs were strongly associated with regulation of transcription, Axon guidance, Pathways in cancer and MAPK signaling pathway. Among these miRNAs, miR-217-5p was the most downregulated miRNA. Sal treatment alleviated cell injury and reduced the production of pro-inflammatory cytokines. Whereas overexpression of miR-217-5p reversed the effects of Sal. We identified YY1 associated factor 2 (YAF2) as a direct target gene of miR-217-5p and Sal treatment could upregulate YAF2 expression via targeting miR-217-5p. Furthermore, knockdown of YAF2 counteracted Sal-induced alleviation of cell injury and inflammation. Moreover, Sal could suppress the activation of p38 MAPK pathway by regulating miR-217-5p/YAF2 axis. ConclusionsOur findings for the first time highlighted that Sal alleviated pancreatic injury and inhibited inflammation by regulating miR-217-5p/YAF2 axis, which might provide new therapeutic strategies for SAP treatment.

Spatial oriented single-cell proteomic assay of low grade serous ovarian carcinoma identifies tumor oncogenic pathways associated with brain metastases (233)

Objectives: As the systemic treatment of epithelial ovarian cancer (EOC) has improved, and the survival is longer, the incidence of brain metastases (BM) in EOC patients has increased to about 2-6% and invariably results in death. In order to better understand the molecular mechanisms involved in the development of EOC BM and to characterize the BM architecture and heterogeneity, we recently used multiplex cyclic-immunofluorescence (Cyc-IF) to characterize a set of matched samples from a single ovarian cancer patient. Methods: The samples included a primary tumor, a peritoneal tumor collected at recurrence, and a BM. Cyc-IF is a spatial oriented single-cell proteomic assay that allows the quantification of over 60 proteins in a single FFPE slide. Results: Our analyses indicated that tumor architecture and heterogeneity were highly altered in the BM compared to the primary and recurrent tumors in this patient. Indeed, the primary and recurrent intraperitoneal tumors showed extensive heterogeneity with cancer cells in different cell states related to the cell cycle (cyclins), hormone receptors (PR and ER) and signaling pathways. In contrast, the BM was overall more homogeneous, with no PR receptor expression and low expression of cyclins. EOC cells from the BM harbor higher oncogenic MAPK and PI3K-AKT signaling pathway activity, higher estrogen receptor expression and displayed immunosuppressive properties including increased expression of PD1 and the presence of tumor-associated macrophages (TAM) and regulatory T cells (Tregs). Importantly, we were able to identify cell populations in the primary tumor that had phenotypes similar to that of cells found in the BM. Conclusions: These findings are consistent with the concept that EOC cells with a predilection for BM are present early during the disease course. Therefore, this represents an opportunity to develop a molecular signature for BM prevention and treatment. Objectives: As the systemic treatment of epithelial ovarian cancer (EOC) has improved, and the survival is longer, the incidence of brain metastases (BM) in EOC patients has increased to about 2-6% and invariably results in death. In order to better understand the molecular mechanisms involved in the development of EOC BM and to characterize the BM architecture and heterogeneity, we recently used multiplex cyclic-immunofluorescence (Cyc-IF) to characterize a set of matched samples from a single ovarian cancer patient. Methods: The samples included a primary tumor, a peritoneal tumor collected at recurrence, and a BM. Cyc-IF is a spatial oriented single-cell proteomic assay that allows the quantification of over 60 proteins in a single FFPE slide. Results: Our analyses indicated that tumor architecture and heterogeneity were highly altered in the BM compared to the primary and recurrent tumors in this patient. Indeed, the primary and recurrent intraperitoneal tumors showed extensive heterogeneity with cancer cells in different cell states related to the cell cycle (cyclins), hormone receptors (PR and ER) and signaling pathways. In contrast, the BM was overall more homogeneous, with no PR receptor expression and low expression of cyclins. EOC cells from the BM harbor higher oncogenic MAPK and PI3K-AKT signaling pathway activity, higher estrogen receptor expression and displayed immunosuppressive properties including increased expression of PD1 and the presence of tumor-associated macrophages (TAM) and regulatory T cells (Tregs). Importantly, we were able to identify cell populations in the primary tumor that had phenotypes similar to that of cells found in the BM. Conclusions: These findings are consistent with the concept that EOC cells with a predilection for BM are present early during the disease course. Therefore, this represents an opportunity to develop a molecular signature for BM prevention and treatment.

Cannabidiol Inhibits Inflammation Induced by Cutibacterium acnes-Derived Extracellular Vesicles via Activation of CB2 Receptor in Keratinocytes

BackgroundAcne is a common inflammatory skin disease, while cannabidiol (CBD) is a representative non-psychoactive phytocannabinoid which has been proved to exert universal anti-inflammatory properties. This study aimed to explore the effect of CBD on acne inflammation induced by Cutibacterium acnes-derived extracellular vesicles (CEVs) in keratinocytes and reveal the underlying mechanisms.MethodsNormal human epidermal keratinocytes (NHEKs) were stimulated by CEVs in the presence of CBD or vehicle. Interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α levels were examined by RT-PCR and ELISA. The expression of cannabinoid type-2 (CB2) receptor and transient receptor potential vanilloid type-1 (TRPV1) was detected by Western blotting. TNF-α levels in the presence of CB2 receptor antagonist (AM630) or TRPV1 antagonist (Capsazepine) were detected by RT-PCR. The activation of MAPK and NF-κB signaling pathways and the nuclear translocation of NF-κB p65 upon CBD treatment were analyzed by Western blotting and immunofluorescence assay, respectively.ResultsThe expression of inflammatory cytokines (IL-6, IL-8 and TNF-α) in CEVs-stimulated NHEKs was suppressed by CBD. CB2 receptor expression was upregulated by CBD, whereas CEVs-promoted TRPV1 expression was downregulated by CBD. AM630 reversed TNF-α levels inhibited by CBD. Capsazepine exerted an inhibitory effect on CEVs-induced inflammation and had synergistic effect with CBD. The phosphorylation of ERK1/2 and NF-κB p65 and nuclear translocation of NF-κB p65 were induced by CEVs but reduced by CBD.ConclusionThe results indicated that CBD could inhibit inflammation induced by CEVs in NHEKs, which was mediated by activation of CB2 receptor and enhanced by the TRPV1 antagonist, through inactivation of the MAPK and NF-κB signaling pathways. CBD might be a potential novel strategy for acne treatment in the future.

Nitidine Chloride Alleviates Inflammation and Cellular Senescence in Murine Osteoarthritis Through Scavenging ROS.

Osteoarthritis (OA) is one of the most common chronic musculoskeletal disorder worldwide, representing a major source of disability, pain and socioeconomic burden. Yet the effective pharmaceutical treatments applied in the clinical works are merely symptomatic management with uncertainty around their long-term safety and efficacy, namely no drugs currently are capable of modulating the biological progression of OA. Here, we identified the potent anti-inflammatory as well as anti-oxidative properties of Nitidine Chloride (NitC), a bioactive phytochemical alkaloid extracted from natural herbs, in IL-1β-treated rat articular chondrocytes (RACs), LPS-stimulated RAW 264.7 and rat osteoarthritic models in vivo. We demonstrated NitC remarkably inhibited the production of inflammatory mediators including COX2 and iNOS, suppressed the activation of MAPK and NF-κB cell signaling pathway and reduced the expression of extracellular matrix (ECM) degrading enzymes including MMP3, MMP9 and MMP13 in IL-1β-treated RACs. Several emerging bioinformatics tools were performed to predict the underlying mechanism, the result of which indicated the potential reactive oxygen species (ROS) clearance potential of NitC. Further, NitC exhibited its anti-oxidative potential through ameliorating cellular senescence in IL-1β-treated RACs and decreasing NLRP3 inflammasomes activation in LPS-stimulated RAW 264.7 via scavenging ROS. Additionally, X-ray, micro-CT and other experiments in vivo demonstrated that intra-articular injection of NitC significantly alleviated the cartilage erosion, ECM degradation and subchondral alterations in OA progression. In conclusion, the present study reported the potent anti-inflammatory and anti-oxidative potential of NitC in OA biological process, providing a promising therapeutic agent for OA management.

080 Current treatment options for patients with unresectable cutaneous melanoma

Cutaneous Melanoma (CM) remains the leading cause of skin-related mortality in the U.S. The U.S. Surveillance, Epidemiology and End Results database reports 5.6% of all new cancer cases in 2021 attributed to CM, with incidences rising annually since 2009. Several subtypes of CM are thought to arise from early driver mutations in BRAF, RAS, and NF1 genes resulting in unopposed activation of MAPK and PI3K signaling pathways, unregulated growth and malignant transformation of melanocytes. Due to the metastatic potential of CM, emphasis on systemic treatment has been an area of interest when surgical resection is insufficient for curative prognosis.

Application of emetine in SARS-CoV-2 treatment: regulation of p38 MAPK signaling pathway for preventing emetine-induced cardiac complications

ABSTRACT Emetine is one of the most highly potent anti-SARS-CoV-2 agents ever identified. In addition to having strong anti-SARS-CoV-2 activities, emetine has other valuable therapeutic effects such as strong anti-inflammatory and anti-arterial pulmonary hypertension (APH) properties, which are suitable for the treatment of COVID-19. Its proper concomitant therapeutic effect has led researchers to test this compound in clinical trials to combat COVID-19. However, due to the risks of cardiac complications, very low doses of emetine have been used in different studies, which may not have significant therapeutic effects. The p38 MAPK signaling pathway is strongly highlighted as an important operator in cardiac cellular damages such as disruption of cardiac fibroblast function and myopathy/cardiomyopathy. Inhibition of this pathway by appropriate inhibitors has also been considered by scientists as a promising strategy for the treatment of fatal host-related hyper-inflammatory immune responses following SARS-CoV-2 infection. Although remarkable stimulatory effects of emetine on activation of the p38 MAPK pathway have been reported in recent studies and strong evidence suggests that this pathway plays an effective role in the emetine’s toxicities, it has not been discussed yet that emetine induced cellular cardiac complications may be due to the activation of this critical pathway. Considering these points could lead to the finding of strategies for applying the valuable potential of emetine in the treatment of COVID-19 at low risks.

Anti-atopic dermatitis effect of a modified Huang-Lian-Jie-Du decoction and its active fraction on 2,4-dinitrobenzene and MC903-induced mouse models

BackgroundHuang-Lian-Jie-Du Decoction is a traditional Chinese medicine formula which has long been used to treat inflammatory skin disease including AD. However, Gardeniae Fructus, a component herb of HLJDD, has noticeable toxicity in liver and kidney. We therefore replaced Gardeniae Fructus with Dictamni Cortex with a hope to derive at a modified HLJDD (MHLJDD) with better safety profile. PurposeThe present study aimed to develop MHLJDD and identify its active fraction as innovative therapeutic agents for AD using 2,4-dinitrobenzene (DNCB) and calcipotriol (MC903)-sensitized mouse models of AD. MethodsMHLJDD and the combination of the 1-butanol-soluble-fraction and the water-soluble-fraction (MHLJDD-F) were given intragastrically to the DNCB-induced mice and MC903-induced mice for two weeks. The body weight, dorsal skin/ear thickness and severity of AD symptoms of the mice were measured throughout the study. Scratching behaviors were observed after drug treatment. The blood and dorsal skin/ear tissues of mice were harvested for histopathological examination and biochemical analyses. ResultsThe results revealed that DNCB- and MC903-induced AD symptoms, including skin thickening, dryness, erythema and excoriations, in the dorsal skin and ears were significantly alleviated in the MHLJDD and MHLJDD-F-treated mice. Ceramides content and protein expressions of filaggrin and loricrin were also up-regulated after treatment with MHLJDD and MHLJDD-F. In addition, skin inflammation induced by DNCB and MC903 were markedly suppressed in the MHLJDD and MHLJDD-F-treated mice, and the action mechanisms involve suppression of the release of inflammatory cytokines, as well as downregulation of the activation of NF-κB and MAPKs pathways. Besides, MHLJDD and MHLJDD-F could reverse the abundance of gut microbiota induced by DNCB in mice. ConclusionsMHLJDD and MHLJDD-F could markedly relieve AD-like symptoms induced by DNCB and MC903 in mice through, at least in part, improving the epidermal barrier function and inhibiting skin inflammation via suppressing the activation of NF-κB and MAPKs pathways and regulation of the gut microflora dysbiosis. This study reported for the first time that MHLJDD and its active fraction could be used as innovative therapeutic agents for AD.

SIRT3 inhibitor 3-TYP exacerbates thioacetamide-induced hepatic injury in mice.

The purpose of the study was to explore the effects of SIRT3 inhibitor 3-TYP on acute liver failure (ALF) in mice and its underlying mechanism. The mice were treated with thioacetamide (TAA, 300 mg/kg) for inducing ALF model. 3-TYP (50 mg/kg) was administered 2 h prior to TAA. The liver histological changes were measured by HE staining. Blood samples were collected for analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). MDA and GSH were used to evaluate the oxidative stress of liver. The expression levels of inflammatory cytokines (TNF-α and IL-1β) were measured by ELISA and Western blotting. The cell type expression of IL-1β in liver tissue was detected by immunofluorescent staining. The expression of SIRT3, MnSOD, ALDH2, MAPK, NF-κB, Nrf2/HO-1, p-elF2α/CHOP, and cleaved caspase 3 was determined by Western blotting. TUNEL staining was performed to detect the apoptosis cells of liver tissues. 3-TYP exacerbated the liver injury of ALF mice. 3-TYP increased the inflammatory responses and activation of MAPK and NF-κB pathways. In addition, 3-TYP administration enhanced the damage of oxidative stress, endoplasmic reticulum stress, and promoted hepatocyte apoptosis in ALF mice. 3-TYP exacerbates thioacetamide-induced hepatic injury in mice. Activation of SIRT3 could be a promising target for the treatment of ALF.

Preclinical Assessment of MEK Inhibitors for Malignant Peripheral Nerve Sheath Tumors Reveals Differences in Efficacy and Adaptive Response.

Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft-tissue sarcomas refractory to standard therapies. Inactivation of NF1 and subsequent upregulation of RAS/RAF/MEK/ERK signaling exist in the majority of MPNSTs. However, the lack of preclinical assessment of MEK inhibitors in MPNSTs hinders the clinical application as well as the development of combination therapy. To guide further clinical studies, we evaluated different MEK inhibitors in terms of efficacy, safety, and mechanism of adaptive response in treating MPNSTs. Using a MPNST tissue microarray, we found that p-ERK could serve as a biomarker for predicting the prognosis of MPNST patients as well as an effective therapeutic target. Through in vitro and in vivo experiments, we identified trametinib as the most potent MEK inhibitor for the treatment of MPNSTs. Mechanistically, reduced reactivation of the MAPK pathway and compensatory activation of the parallel pathways contributed to better efficacy. Our results provide a basis for the further clinical application of MEK inhibitors as single agents or combinational therapies.

Cepharanthine Ameliorates Chondrocytic Inflammation and Osteoarthritis via Regulating the MAPK/NF-κB-Autophagy Pathway.

Osteoarthritis is a worldwide joint disease caused by abnormal chondrocytic metabolism. However, traditional therapeutic methods aimed at anti-inflammation for early-stage disease are palliative. In the present study, we demonstrated that cepharanthine (CEP), extracted from the plant Stephania cepharantha, exerted protective medicinal efficacy on osteoarthritis for the first time. In our in vitro study, CEP suppressed the elevated expression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and inducible nitric oxide synthase (iNOS) stimulated by IL-1β or TNF-α by inhibiting the activation of MAPK and NF-κB signaling pathways, and upregulated the protein expression of aggrecan, collagen II, and Sox9. Also, CEP could reverse the reduced level of cellular autophagy in IL-1β or TNF-α–induced chondrocytes, indicating that the protective effect of CEP on osteoarthritis was achieved by restoring MAPK/NF-κB-mediated autophagy. Furthermore, in a murine OA model, CEP mitigated cartilage degradation and prevented osteoarthritis in the CEP-treated groups versus the OA group. Hence, our results revealed the therapeutic prospect of CEP for anti-osteoarthritic treatment.

Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

BackgroundMedulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive.MethodsBy taking advantage of Math1-Cre/Ptch1loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo.ResultsWe found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo.ConclusionsC3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.

Abstract 1127: Effect of the combined therapy of sotorasib and metformin in non-small cell lung cancer cell lines

Abstract KRAS is the most frequently mutated oncogene in lung adenocarcinoma, in which is related to poor treatments response and worse survival rates than other driver mutations. KRAS activates MAPK and mTOR pathways, and even after sotorasib treatment, a novel KRAS inhibitor, the duration of responses to this monotherapy are excessively short. In this context, metformin is able to inhibit PI3K and mTOR pathways; therefore, combined use of metformin with sotorasib aids to diminish the activity of both signaling pathways. We performed a dose-effect curve to determine IC50 of sotorasib, and IC10 of metformin to treat lung cancer cell lines harboring different mutational profiles: A549 (KRAS G12S), and H522 (TP53) in order to study the effects of the combination compared to the monotherapy of each drug at MTT assay and western blot analysis. In detail, we evaluated drug-induced cytotoxicity, apoptosis, and activity of MAPK and mTOR pathways. Our results show a synergic effect in cytotoxicity by MTT assay, as well as increased inhibition of MAPK pathway inhibition at combinational treatment, compared to monotherapies in A549 cell lines; conversely, we did not find significant effects on cytotoxicity and signaling inhibition in H522 cell line. In conclusion, the combined treatment of metformin and sotorasib show synergistic effects in drug-induced cytotoxicity and inhibition of MAPK pathways in lung cancer cell lines. Citation Format: Norma Yanet Hernandez-Pedro, Jose Lucio-Lozada, Pedro Barrios-Bernal, Maritza Ramos-Ramírez, Oscar Arrieta. Effect of the combined therapy of sotorasib and metformin in non-small cell lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1127.

Abstract 87: EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma

Abstract Background: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R) has been implicated in EWS tumorigenesis and development of metastatic disease, with anti-IGF1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF1R-driven oncogenesis in EWS could provide avenues for more effective therapy. Intracellular trafficking plays a central role in regulating RTK expression and signaling, and this pathway is frequently deregulated in cancer cells. The EPS15 homology domain-containing (EHD) proteins regulate intracellular traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Study design: Three EHD1-expressing EWS cell lines, TC71, SKES1, and A673, were engineered with doxycycline-inducible EHD1 shRNAs to assess the impact of EHD1 knockdown (KD) on in vitro oncogenic properties. Stable EHD1-Crispr-KO and mouse EHD1-rescue TC71 and A673 cell line models were developed and characterized. Further, non-targeting control or EHD1-KD TC71 cells engineered with RFP-luciferase reporter were implanted in the tibia of nude mice in an orthotopic xenograft model and tumor growth was monitored by IVIS imaging. Results: EHD1-KD led to a significant impairment of EWS cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation in vitro and reduced tumor growth in nude mice. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF1R levels upon EHD1-KD, identifying IGF1R as a potential target of regulation by EHD1. Western blotting showed a reduction in total IGF1R levels, and flow cytometric and immunofluorescence analyses revealed a pronounced decrease in the cell surface IGF1R levels upon EHD1 KD/KO in EWS cell lines. EHD1-KO EWS cell lines also exhibited a defect in IGF1-dependent migration and proliferation, and the phenotypes were restored by mouse EHD1 rescue. IGF1R and EHD1 were found to colocalize intracellularly and to co-immunoprecipitate after IGF1 stimulation. Finally, EHD1 depletion was found to impair the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Conclusion: Our studies indicate a novel requirement of the intracellular traffic regulator EHD1 in sustaining IGF1R mediated oncogenesis in EWS. Future studies will aim to evaluate dual targeting of EHD1 and IGF1R in EWS. Citation Format: Sukanya Chakraborty, Bhopal C. Mohapatra, Sameer Mirza, Aaqib M. Bhat, Matthew D. Storck, Isidro Machado, José Antonio López, Antonio Llomart Bosch, Donald W. Coulter, Gargi Ghosal, Vimla Band, Hamid Band. EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 87.

Abstract 48: Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC)

Abstract Background: Alterations in the RAS pathway have been linked to tumorigenesis, apoptosis, metabolism and angiogenesis. Mutations of KRAS in NSCLC are more frequent and better characterized, however, other family members such as HRAS remain under investigated and RAS remains a challenging therapeutic target. HRAS has been indirectly targeted with tipifarnib, a farnesyltransferase inhibitor rendering HRAS inactive in head and neck tumors. Here, we characterize the incidence, genomic landscape, and clinical context of HRAS alterations in NSCLC. Methods: A total of 29,767 NSCLC tumor samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. MAPK activation was assessed using the MPAS gene expression signature. Wilcoxon, Fisher’s exact, or Dunnett’s tests were used to determined statistical significance (displayed as p value without and q value with multi-comparison correction). Overall survival was calculated from date of tissue collection to last contact from insurance claims data and used for Kaplan-Meier analysis. Comparisons were conducted between HRAS mutated tumors and the entire NSCLC general cohort (GC). Results: HRAS mutations (Hm) were detected in 128 of 29767 NSCLC samples (0.4%) and were significantly enriched in older patients (median age, 71 vs. 69 years; q<0.01) and squamous histology (57.8% vs 21.8%, q<0.0001) compared to GC. Smoking status was not associated with HRAS mutational status (p=0.19). The most prevalent loci of hotspot mutations in Hm tumors were G13 (42.2%), followed by Q61 (33.6%) and G12 (21.1%). HRAS was not prognostic for overall survival (HR = 1.06, 95% CI [0.83-1.35], p=0.64), but for HRAS G13 mutant subset of NSCLC there was a trend towards worse prognosis (HR = 1.31, 95% CI [0.91-1.88], p =0.14). Hm-positive tumors harbored significantly more PIK3CA mutations (16.9% vs 5.5%, q<0.05) but less KRAS (3.2% vs 27.4%, q<0.05) and EGFR mutations (0.8% vs 11.9%, q<0.05) compared to GC. In addition, Hm displayed dMMR/MSI deficiency (2.6% vs 0.7%, p <0.05) more frequently, but had a comparable percentage of TMB-H tumors (34.2% vs 40.2%, p = 0.16) and similar median PD-L1 expression (54.7% vs 60.3%, p = 0.21) when comparing to GC. Lastly, MAPK pathway activation score was relatively higher in Hm compared to GC (-0.26 vs -0.42) with Q61 being the highest group (0.04 vs -0.42, p = 0.37). Conclusions: HRAS mutations are detectable but uncommon events in NSCLC and significantly enriched in squamous histology. HRAS mutations often occur with PIK3CA co-mutations and trended towards higher activation of MAPK pathway and MSI-H frequency. This warrants further investigation on possible clinical applications of HRAS pathway inhibitors and utility of immune checkpoint inhibitors for this subset of NSCLC. Citation Format: Asaad Trabolsi, Estelamari Rodriguez, Samuel Alexander Kareff, Michael Korn, Joanne Xiu, Stephen Liu, Philip walker, Patrick Ma, Hirva Mamdani, Jorge Nieva, Hossein Borghaei, Chadi Nabhan, Misako Nagasaka, Sonam puri, Gilberto Lopes. Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 48.