Tumor Growth In Xenograft Model Research Articles

A Sampling of Highlights from the Literature

T cells expressing WT137–45-specific TCRs kill AML cells (by BruceBlaus via Wikimedia Commons)T cells engineered to express T-cell receptors (TCR) targeting intracellular tumor antigens have potential for treating cancer. Two studies identify HLA-A*02:01–restricted TCRs specific for Wilms’ tumor antigen 1 (WT1) peptide WT137–45 as candidates for clinical translation. Ruggiero et al. show that T cells engineered to express such TCRs significantly reduce tumor growth in xenograft models of acute myeloid leukemia (AML), acute lymphoblastic leukemia, and glioblastoma. Lahman et al. show that generation of WT137–45 is immunoproteasome independent, which is important because AML recurrence in one patient they treated with T cells expressing an HLA-A*02:01–restricted, WT126–134-specific TCR occurred because of loss of immunoproteasome processing of WT1.Ruggiero E, …, Bonini C. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8027.Lahman MC, …, Greenberg PD. Sci Trans Med 2022 February 9;14. DOI:10.1126/scitranslmed.abg8070.PERK signaling promotes M2 macrophage immunosuppressive function (by Noahtsmith via Wikimedia Commons)Intratumoral macrophage phenotype is determined by the signals the cells receive from the tumor microenvironment. Raines et al. identify new molecular pathways that promote the activation and immunosuppressive function of M2 macrophages. They find that tumor-associated macrophages from patients with lung carcinoma are characterized by increased activity of protein kinase RNA-like ER kinase (PERK) signaling. This promotes an immunosuppressive phenotype in macrophages through metabolic reprograming and histone demethylation. Inhibition of PERK signaling reduces growth of B16-F10 tumors. This is associated with reduced numbers and immunosuppressive activity of intratumoral macrophages, suggesting PERK signaling inhibition as a potential therapeutic approach to target macrophages.Raines LN, …, Huang SCC. Nature Immunol 2022;23:431–445.CRISPR technology allows for characterization of T-cell functional regulators (by Clipart Panda via Clipart Panda)Identifying regulators of cytokine production upon cellular activation has had its limitations. Using novel CRISPR screens, Schmidt et al. identify regulators of IL2 and IFNγ production by T cells. Distinct cytokine profiles are identified and characterize T cells during various states of activation. The data highlight screening that can be performed to determine how T cells can be reprogramed and could be used to better design immunotherapy for the treatment of cancer.Schmidt R, …, Marson A. Science 2022 February 4;375. DOI:10.1126/science.abj4008.Prediction modeling could be used to identify patients who will respond to immune checkpoint blockade (by Eva K. via Wikimedia Commons)Identifying markers of therapy response and patient outcome is an unmet clinical need. Patil et al. find that plasma cells in tumoral tertiary lymphoid structures are a biomarker of overall survival in patients treated with immune checkpoint blockade (ICB), an effect independent of tumor CD8+ T cells and PD-L1 expression. Asrir et al. identify tumor-associated high endothelial venules as key players for lymphocyte entry into tumors and associated with survival and response to combination ICB. These two studies highlight key immune mechanisms and biomarkers that could be potentially used to improve treatment strategies for patients with cancer.Asrir A, …, Girard JP. Cancer Cell 2022 March 14;40:318–334.e9.Patil NS, …, Shames DS. Cancer Cell 2022 March 14;40:289–300.e4.CAR T cells are being developed for many types of cancer (from Fig. 7B of Kantari-Mimoun et al., Cancer Immunol Res 2021)Patient data following chimeric-antigen receptor (CAR) T-cell therapy are critical to ongoing clinical development. Through longitudinal analysis of two patients with chronic lymphocytic leukemia who achieved lasting complete remission after CD19-specific CAR T-cell therapy in 2010, Melenhorst et al. find that CAR T cells persist for more than a decade, with a population of highly activated CD4+ CAR T cells dominating in both patients. Majzner et al. report that three of four patients with H3K27M-mutant diffuse midline glioma who received GD2-specific CAR T cells intravenously and then intracerebroventricularly had clinical and radiographic improvement. These studies provide new insights that will inform continued CAR T-cell development for hematological malignancies and solid tumors.Melenhorst JJ, …, June CH. Nature 2022;602:503–509.Majzner RG, …, Monje M. Nature 2022 February 7;14. DOI:10.1038/s41586-022-04489-4.The mycobiome facilitates pancreatic tumor progression (Pancreas, in Opera Omnia via Wellcome Collection)T helper 2 cells (TH2) and innate lymphoid cells 2 (ILC2) infiltrate pancreatic ductal adenocarcinomas (PDAC) and secrete cytokines that promote tumorigenesis. Alam et al. identify KrasG12D-regulated IL33 as a mediator of TH2 and ILC2 recruitment in mouse models of PDAC. Fungal species found in the gut and tumor microenvironment induce tumor cell IL33 secretion by activating the dectin-1 receptor and downstream Src–Syk–CARD9 pathway. Anti-fungal treatment reduces TH2 and ILC2 infiltration and reduces tumor growth, highlighting a novel mechanism by which the PDAC tumor mycobiome can regulate the pro-tumorigenic inflammatory response and suggesting a potential therapeutic strategy.Alam A, …, Dey P. Cancer Cell 2022 February 14;40:153–167.E11.

Resistance to obinutuzumab-induced antibody-dependent cellular cytotoxicity caused by abnormal Fas signaling is overcome by combination therapies

BackgroundObinutuzumab, a Type II anti-CD20 antibody, is used to treat follicular lymphoma. A major mode of action of obinutuzumab is antibody-dependent cellular cytotoxicity (ADCC). Knowledge of the mechanisms of resistance to obinutuzumab is important for the development of next-line strategies to follow obinutuzumab-containing therapy, including obinutuzumab retreatment. Unfortunately, the mechanisms by which tumor cells acquire resistance to ADCC are still poorly understood. To address this, we examined the mechanisms of resistance to obinutuzumab-induced ADCC and the combination efficacy of obinutuzumab and clinically available agents in the established resistant cells.Methods and resultsWe established cells resistant to obinutuzumab-induced ADCC using the non-Hodgkin lymphoma cell line RL and examined their mechanisms of resistance and the combination efficacy of obinutuzumab and clinically available agents. Comprehensive analysis by RNA sequencing of resistance mechanisms revealed that abnormal Fas signaling decreased sensitivity to ADCC in resistant clones. Combination treatment with prednisolone, a component of CHOP and CVP, was found to enhance ADCC sensitivity of RL cells and resistant clones and to significantly suppress tumor growth in xenograft models. Treatment with prednisolone upregulated expression of CD20 and an apoptosis-inducing protein BIM, which might augment perforin/granzyme B-mediated cell death. Furthermore, pretreatment of the effector cells with bendamustine enhanced ADCC activity, and treatment with obinutuzumab plus bendamustine showed significant antitumor efficacy in xenograft models. It was speculated that bendamustine upregulates ADCC activity by potentiating granules-mediated cell killing.ConclusionsOur study revealed a novel mechanism underlying obinutuzumab-induced ADCC resistance and indicated that ADCC resistance could be overcome by combining obinutuzumab with prednisolone or bendamustine. This study provides a scientific rationale for obinutuzumab-retreatment in combination with clinically available chemotherapeutic agents for obinutuzumab resistant follicular lymphoma.

The phosphatase CTDSPL2 is phosphorylated in mitosis and a target for restraining tumor growth and motility in pancreatic cancer.

Carboxy-terminal domain (CTD) small phosphatase like 2 (CTDSPL2), also known as SCP4 or HSPC129, is a new member of the small CTD phosphatase (SCP) family and its role in cancers remains unclear. Here, we used a Phos-tag technique to screen a series of phosphatases and identified CTDSPL2 as a mitotic regulator. We demonstrated that CTDSPL2 was phosphorylated at T86, S104, and S134 by cyclin-dependent kinase 1 (CDK1) in mitosis. Depletion of CTDSPL2 led to mitotic defects and prolonged mitosis. Resultantly, CTDSPL2 deletion restrained proliferation, migration, and invasion in pancreatic cancer cells. We further confirmed the dominant negative effects of a phosphorylation-deficient mutant form of CTDSPL2, implying the biological significance of CTDSPL2 mitotic phosphorylation. Moreover, RT2 cell cycle array analysis revealed p21 and p27 as downstream regulators of CTDSPL2, and inhibition of p21 and/or p27 partially rescued the phenotype in CTDSPL2-deficient cell lines. Importantly, both CTDSPL2 depletion and phosphorylation-deficient mutant CTDSPL2 hindered tumor growth in xenograft models. Together, our findings for the first time highlight the novel role of CTDSPL2 in regulating cell mitosis, proliferation and motility in pancreatic cancer and point out the implications of CTDSPL2 in regulating two critical cell cycle participants (p21 and p27), providing an alternative molecular target for pancreatic cancer treatment.

Green tea-derived theabrownin induces cellular senescence and apoptosis of hepatocellular carcinoma through p53 signaling activation and bypassed JNK signaling suppression

BackgroundTheabrownin (TB) is a bioactive component of tea and has been reported to exert effects against many human cancers, but its efficacy and mechanism on hepatocellular carcinoma (HCC) with different p53 genotypes remains unclarified.MethodsMTT assay, DAPI staining, flow cytometry and SA-β-gal staining were applied to evaluate the effects of TB on HCC cells. Quantitative real time PCR (qPCR) and Western blot (WB) were conducted to explore the molecular mechanism of TB. A xenograft model of zebrafish was established to evaluate the anti-tumor effect of TB.ResultsMTT assays showed that TB significantly inhibited the proliferation of SK-Hep-1, HepG2, and Huh7 cells in a dose-dependent manner, of which SK-Hep-1 was the most sensitive one with the lowest IC50 values. The animal data showed that TB remarkably suppressed SK-Hep-1 tumor growth in xenograft model of zebrafish. The cellular data showed TB's pro-apoptotic and pro-senescent effect on SK-Hep-1 cells. The molecular results revealed the mechanism of TB that p53 signaling pathway (p-ATM, p-ATR, γ-H2AX, p-Chk2, and p-p53) was activated with up-regulation of downstream senescent genes (P16, P21, IL-6 and IL-8) as well as apoptotic genes (Bim, Bax and PUMA) and proteins (Bax, c-Casp9 and c-PARP). The p53-mediated mechanism was verified by using p53-siRNA. Moreover, by using JNK-siRNA, we found JNK as a bypass regulator in TB's mechanism.ConclusionsTo sum up, TB exerted tumor-inhibitory, pro-senescent and pro-apoptotic effects on SK-Hep-1 cells through ATM-Chk2-p53 signaling axis in accompany with JNK bypass regulation. This is the first report on the pro-senescent effect and multi-target (p53 and JNK) mechanism of TB on HCC cells, providing new insights into the underlying mechanisms of TB's anti-HCC efficacy.

Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

SPDEF suppresses head and neck squamous cell carcinoma progression by transcriptionally activating NR4A1

SAM pointed domain containing E26 transformation-specific transcription factor (SPDEF) plays dual roles in the initiation and development of human malignancies. However, the biological role of SPDEF in head and neck squamous cell carcinoma (HNSCC) remains unclear. In this study, the expression level of SPDEF and its correlation with the clinical parameters of patients with HNSCC were determined using TCGA-HNSC, GSE65858, and our own clinical cohorts. CCK8, colony formation, cell cycle analysis, and a xenograft tumor growth model were used to determine the molecular functions of SPDEF in HNSCC. ChIP-qPCR, dual luciferase reporter assay, and rescue experiments were conducted to explore the potential molecular mechanism of SPDEF in HNSCC. Compared with normal epithelial tissues, SPDEF was significantly downregulated in HNSCC tissues. Patients with HNSCC with low SPDEF mRNA levels exhibited poor clinical outcomes. Restoring SPDEF inhibited HNSCC cell viability and colony formation and induced G0/G1 cell cycle arrest, while silencing SPDEF promoted cell proliferation in vitro. The xenograft tumor growth model showed that tumors with SPDEF overexpression had slower growth rates, smaller volumes, and lower weights. SPDEF could directly bind to the promoter region of NR4A1 and promoted its transcription, inducing the suppression of AKT, MAPK, and NF-κB signaling pathways. Moreover, silencing NR4A1 blocked the suppressive effect of SPDEF in HNSCC cells. Here, we demonstrate that SPDEF acts as a tumor suppressor by transcriptionally activating NR4A1 in HNSCC. Our findings provide novel insights into the molecular mechanism of SPDEF in tumorigenesis and a novel potential therapeutic target for HNSCC.

The RNA N6-Methyladenosine Methyltransferase METTL3 Promotes the Progression of Kidney Cancer via N6-Methyladenosine-Dependent Translational Enhancement of ABCD1.

The role of N6-methyladenosine (m6A)-modifying proteins in cancer progression depends on the cell type and mRNA affected. However, the biological role and underlying mechanism of m6A in kidney cancer is limited. Here, we discovered the variability in m6A methyltransferase METTL3 expression was significantly increased in clear cell renal cell carcinoma (ccRCC) the most common subtype of renal cell carcinoma (RCC), and high METTL3 expression predicts poor prognosis in ccRCC patients using a dataset from The Cancer Genome Atlas (TCGA). Importantly, knockdown of METTL3 in ccRCC cell line impaired both cell migration capacity and tumor spheroid formation in soft fibrin gel, a mechanical method for selecting stem-cell-like tumorigenic cells. Consistently, overexpression of METTL3 but not methyltransferase activity mutant METTL3 can promote cell migration, spheroid formation in cell line and tumor growth in xenograft model. Transcriptional profiling of m6A in ccRCC tissues identified the aberrant m6A transcripts were enriched in cancer-related pathways. Further m6A-sequencing of METTL3 knockdown cells and functional studies confirmed that translation of ABCD1, an ATP-binding cassette (ABC) transporter of fatty acids, was inhibited by METTL3 in m6A-dependent manner. Moreover, knockdown of ABCD1 in ccRCC cells decreased cancer cell migration and spheroid formation, and upregulation of ABCD1 acts as an adverse prognosis factor of kidney cancer patients. In summary, our study identifies that METTL3 promotes ccRCC progression through m6A modification-mediated translation of ABCD1, providing an epitranscriptional insight into the molecular mechanism in kidney cancer.

SHARPIN regulates the development of clear cell renal cell carcinoma by promoting von Hippel-Lindau protein ubiquitination and degradation.

SHANK‐associated RH domain interacting protein (SHARPIN) plays an important role in carcinogenesis, as well as inflammation and immunity. Our study explored the effects and underlying mechanisms of SHARPIN in clear cell renal cell carcinoma (ccRCC). By analyzing The Cancer Genome Atlas database, we found that upregulated SHARPIN in patients with ccRCC led to a poor prognosis. Semiquantitative immunohistochemical analysis of clinical samples was carried out and the results suggested the positive association between SHARPIN and hypoxia‐induced factor‐2α (HIF‐2α). Von Hippel‐Lindau protein (pVHL) is a tumor suppressor that contributes to degrading HIF‐2α. Mechanically, SHARPIN promoted the ubiquitination and proteasomal degradation of pVHL, resulting in the sustained activation of HIF‐2α. The α and β domains of pVHL and ubiquitin‐like domain of SHARPIN are required for the interaction. The knockdown of SHARPIN effectively inhibited acquired sorafenib resistance in ccRCC cell lines and tumor growth in xenograft models. In conclusion, our work reveals a novel posttranslational regulation of SHARPIN on pVHL, indicating that SHARPIN could be a potential target for ccRCC treatment.

Water-Soluble Total Flavonoids Isolated from Isodon lophanthoides var. gerardianus (Benth.) H. Hara Promote Hepatocellular Carcinoma Sensitivity to 5-Fluorouracil.

Isodon lophanthoides var. gerardianus (Benth.) H. Hara, a native medicinal plant produced chiefly across Southern China, is one of the mainstream varieties of Xihuangcao, which has long been applied in preventing and treating some common liver or gall diseases. Water-soluble total flavonoids (WSTF) extracted from folk herbal medicine have many pharmacological effects. The objective of the paper is to investigate the synergy of WSTF with 5-fluorouracil (5-FU) on HCC and the related mechanisms. Cells were exposed to WSTF alone or combination treatment with 5-FU. Then, in this study, we conducted cell viability test, cell cycle and clone forming test, apoptosis assay, reactive oxygen species (ROS), Western blotting, immunohistochemistry, and a xenograft tumor growth model for investigating the role of WSTF in HCC in vivo and in vitro. It was discovered that WSTF caused cell cycle arrest at the G0/G1 phase while increasing the ROS contents. The generation of ROS levels could cause cell apoptosis and inhibit colony formation. WSTF decreased the Bcl-2 level but promoted the Bax level. These showed the mitochondrial dependence of WSTF-mediated apoptosis. WSTF combined with 5-FU have a synergistic effect to significantly inhibit carcinogenicity in vivo and in vitro. The reduced ROS changed the synergy of WSTF with 5-FU. At last, WSTF inhibit the growth of HCC and promote the HCC sensitivity to 5-FU through ROS accumulation. WSTF-increased ROS levels may partially or completely contribute to enhanced toxicity. WSTF combined with 5-FU in HCC can play a synergistic effect when applied in the clinical setting.

Abstract 738: Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition

Abstract Introduction: Approximately 70% of human breast cancers express estrogen receptor alpha (ERα), providing the potential for targeted endocrine therapy for patients. Unfortunately, 30-40% of ER+ patients still experience recurrence and metastasis with a 5-year relative overall survival rate of just 24%. Previously, we described metabolic changes in metastatic breast cancer cells that are driven by single therapies and the metastatic organ microenvironment. Combining selinexor (SEL), a small molecule inhibitor of exportin 1 (XPO1) approved by the FDA for treatment of some hematologic malignancies, with the current therapies used for advanced breast cancers (e.g. tamoxifen) decreased metabolic adaptations in response to individual drug treatments and prevented colony formation in hydrogels and tumor growth in xenograft models. Since we observed a reliance on NAD+ metabolism in cancer cells that are treated with single therapies, in the current study we investigated co-targeting metabolic alterations using SEL and the NAMPT inhibitor KPT-9274. Methods: We established 3D cell culture systems in novel hydrogels derived from decellularized bone, lung, and liver tissues - the major sites of metastasis of ER+ breast cancers, and compared with standard Matrigel culture. Endocrine responsive (MCF7 and T47D) and resistant (MCF7 and T47D cells with ESR1 Y537S or D538G mutations) breast cancer cell lines were treated with SEL or KPT-9274. Tumorigenicity was assessed using in vitro cell viability and colony formation assays and in vivo breast cancer cell line xenografts in athymic mice. Metabolic changes were assessed using whole metabolite profiling by GC-MS and a Seahorse XF analyzer. Results: Combining an FDA-approved XPO1 small molecule inhibitor, Selinexor (SEL) with the NAMPT inhibitors decreased metabolic adaptations in response to individual drug treatments, and prevented colony formation in hydrogels and tumor growth in xenograft models. Conclusions: Targeting drug and metastatic-site induced adaptations to regenerate new vulnerabilities in endocrine-resistant breast tumors are novel. Given the need for better strategies for improving therapy response of metastatic ER+ tumors, our findings show uncovering the role XPO1-NAMPT crosstalk plays in metastatic breast cancer could lead to new combined therapies that reduce mortality. Citation Format: Qianying Zuo, Ayca Mogol, Ozan Imir, Yosef Landesman, Zeynep Madak Erdogan. Targeting metabolic vulnerabilities of endocrine resistant breast cancers using XPO1 and NAMPT inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 738.

Abstract 980: Preclinical evaluation of mps1 inhibition in uterine leiomyosarcoma

Abstract Background: Uterine leiomyosarcoma (Ut-LMS) is the commonest malignant mesenchymal tumor arising from the uterus. It is aggressive and the prognosis is dismal. To-date, no specific oncogenic drivers have been identified. Conventional adjuvant therapy and immunotherapy are ineffective and treatment largely remains surgical. MPS1 kinase is vital in spindle assembly checkpoint which safeguards the faithful segregation of chromosomes. Its inactivation leads to mitotic defects and induces mitotic catastrophe. Compare with normal tissue, MPS1 has shown to be overexpressed in Ut-LMS. Method: The Ut-LMS cell lines SK-UT-1 and SKN were treated with a MPS1 inhibitor. RT-qPCR was performed to analyze mRNA expression levels of Spindle Assembly Checkpoint-related genes. Cells were seeded in 96-well plates and treated with MPS1 inhibitor for 4 days. Sulforhodamine B stain was used to labelled viable cells. IC50 of different cell lines was determined by SRB assay. Cell cycle analysis was done after 2 days treatment by propidium iodide staining. Apoptosis study was done after 3 days treatment by AnnexinV/PI apoptotic assay. Flow cytometry was used to analyze PI assay and AnnexinV/PI apoptotic assay. Immunofluorescence staining of centromere, α-tubulin and DAPI was used to label kinetochore, mitotic spindles and DNA. Cells with chromosome missegregation were counted in treated and non-treated samples. Ut-LMS cell lines SKN was injected subcutaneously in Balb/c nude mice and MPS1 inhibitor was administrated through oral gavage daily, tumor weight was record and tumor size was estimated by caliper. Results: The Spindle Assembly Checkpoint-related genes MPS1, CDC20, MAD2L1, BUB1, BUB1B, and KNL1 were found to be overexpressed. SRB assays had shown that there was growth inhibition of both cell lines in a concentration-dependent manner with IC50 values ranged between 40-65 nM. An increase in aneuploidy cells was demonstrated by PI assay, which suggested that MPS1 inhibitor disrupted cell cycle and induced aneuploidy. The inhibitor also induced apoptosis as demonstrated an increase in AnnexinV+/PI+ cells. Chromosome missegregation was demonstrated in double-thymidine block synchronized anaphase cells with immunofluorescence staining. Micronuclei were also identified in tetraploid cells. In a preliminary in vivo study, MPS1 inhibitor was shown to reduce tumor growth in xenograft models. Conclusion: Our preclinical results indicated that MPS1 inhibitor was an effective treatment in Ut-LMS as a single agent by causing chromosome missegregation, mitotic catastrophe, genomic instability and cell death. Further evaluation of its use in combination with other pharmacological agents is in progress. Citation Format: Ho Shing Wong, Hok Yeung H. Lee, Ka Yu Tse, Tak Wah Mak, Philip PC Ip. Preclinical evaluation of mps1 inhibition in uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 980.

LncRNA ITGB2-AS1 promotes the progression of clear cell renal cell carcinoma by modulating miR-328-5p/HMGA1 axis.

Clear cell renal cell carcinoma (ccRCC) is the most common histologic subtype of renal cell carcinoma and long non-coding RNAs (lncRNAs) play important roles in the progression of ccRCC. In this study, we aim to explore the potential function of ITGB2-AS1 in ccRCC progression and its underlying molecular mechanism. We first explored the association between ITGB2-AS1 expression level and ccRCC prognosis. We found that the expression level of ITGB2-AS1 was significantly higher in ccRCC tumor and cell lines, and highly expressed ITGB2-AS1 was also associated with a poorer prognosis. Consistently, silencing ITGB2-AS1 inhibited proliferation, promoted apoptosis in ccRCC cell lines, and curbed the tumorigenesis in the Xenograft model, reduced tumorigenesis in a xenograft tumor growth model. We further identified and confirmed the miRNA miR-328-5p as a target of ITGB2-AS1, and miR-328-5p negatively regulated the expression of HMGA1 protein. The anti-tumor effect of silencing ITGB2-AS1 could be partially rescued by inhibiting miR-328-5p activity or overexpressing HMGA1, indicating that ITGB2-AS1 promotes the survival and progression of ccRCC by modulating miR-328-5p/HMGA1 axis. Collectively, our data demonstrated that ITGB2-AS1 expression level is positively correlated with the survival and tumorigenesis of ccRCC. As a target of ITGB2-AS1, miR-328-5p seems to function as a tumor-suppressor, and the oncogenic effect of ITGB2-AS1 is partially mediated via the miR-328-5p/HMGA1 axis.

BCAT1 Activates PI3K/AKT/mTOR Pathway and Contributes to the Angiogenesis and Tumorigenicity of Gastric Cancer.

BackgroundFocusing on antiangiogenesis may provide promising choices for treatment of gastric cancer (GC). This study aimed to investigate the mechanistic role of BCAT1 in the pathogenesis of GC, particularly in angiogenesis.MethodsBioinformatics and clinical samples analysis were used to investigate the expression and potential mechanism of BCAT1 in GC. BGC823 cells with BCAT1 overexpression or silencing were induced by lentiviral transduction. Cell phenotypes and angiogenesis were evaluated. The relevant proteins were quantized by Western blotting, immunohistochemistry, or immunofluorescence. Xenograft models were constructed to confirm the role of BCAT1 in vivo.ResultsBCAT1 was overexpressed in GC patients and associated with lower survival. BCAT1 expression was correlated with proliferation-, invasion-, or angiogenesis-related markers expression and pathways. Silencing BCAT1 expression suppressed cell viability, colony formation, cycle progression, invasion, and angiogenesis of BGC823 cells, as well as the tumor growth of xenograft models, whereas overexpressing BCAT1 had the opposite results both in vitro and in vivo. Bioinformatics analysis and Western blotting demonstrated that BCAT1 activated the PI3K/AKT/mTOR pathway. The addition of LY294002 reversed the tumor growth induced by BCAT1 overexpression, further verifying this mechanism.ConclusionBCAT1 might act as an oncogene by facilitating proliferation, invasion, and angiogenesis through activation of the PI3K/AKT/mTOR pathway. This finding could aid the optimization of antiangiogenesis strategies.

YAP/TEAD4-induced KIF4A contributes to the progression and worse prognosis of esophageal squamous cell carcinoma.

Aberrant expression of kinesin family member 4A (KIF4A), which is associated with tumor progression, has been reported in several types of cancer. However, its expression and the underlying molecular mechanisms regulating the transcription of KIF4A in esophageal squamous cell carcinoma (ESCC) remain largely unclear. Here, we found that high KIF4A expression was positively correlated with tumor stage and poor prognosis in ESCC patients. KIF4A silencing significantly inhibited the growth and migration of ESCC cells, arrested cell cycle, and induced apoptosis. Interestingly, KIF4A expression was positively related to the expression of YAP in human ESCC tissues. YAP knockdown or disrupting YAP/TEAD4 interaction by verteporfin repressed KIF4A expression. Also, KIF4A knockdown significantly inhibited the cell growth induced by YAP overexpression. Mechanistically, YAP activated KIF4A transcriptional expression by TEAD4-mediated direct binding to KIF4A promoter. Finally, KIF4A knockdown and verteporfin treatment synergistically inhibited tumor growth in xenograft models. Together, these results indicated that KIF4A, a novel target gene of YAP/TEAD4, may be a progression and prognostic biomarker of ESCC. Targeting drugs for KIF4A combined with YAP inhibitor may be a novel therapeutic strategy for ESCC.

Combined Blockade of MEK and CDK4/6 Pathways Induces Senescence to Improve Survival in Pancreatic Ductal Adenocarcinoma.

Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDK) that induce cell-cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition (CDK4/6i) may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared with normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6i results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared with monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared with treatment with vehicle or individual agents alone in Ptf1acre/+ ;LSL-KRASG12D/+ ;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.

CircTET1 Inhibits Retinoblastoma Progression via Targeting miR-492 and miR-494-3p through Wnt/β-catenin Signaling Pathway

ABSTRACT Purpose: Retinoblastoma (RB) is a frequent intraocular malignancy in children. Circular RNA (circRNA) plays an essential role in regulating the occurrence and development of tumors. This study aimed at investigating the function and molecular basis of hsa_circ_0093996 (circTET1) in RB. Methods: The expression of circTET1, miR-492 and miR-494-3p was examined using quantitative real-time polymerase chain reaction. Cell proliferation, cycle arrest, apoptosis, migration and invasion of RB cells were detected using Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, scratch assay and transwell analysis, respectively. The levels of matrix metalloproteinase (MMP) 2, MMP9 and Wnt/β-catenin pathway-related proteins were measured via western blot assay. The association between circTET1 and miR-492/miR-494-3p was validated via dual-luciferase reporter assay and RNA pull-down assay. Xenograft assay was employed to analyze tumor growth in vivo. Results: CircTET1 level was reduced, while miR-492 and miR-494-3p levels were increased in RB tissues and cells. Overexpression of circTET1 inhibited proliferation, migration and invasion, and promoted apoptosis and cell cycle arrest in Y79 and WERI-Rb1 cells. Moreover, circTET1 impeded RB cell progression by sponging miR-492/miR-494-3p. Also, up-regulation of circTET1 restrained Wnt/β-catenin pathway via regulating miR-492 and miR-494-3p. Furthermore, circTET1 suppressed tumor growth in xenograft models. Conclusion: CircTET1 inhibited RB progression by sponging miR-492/miR-494-3p and inactivating the Wnt/β-catenin pathway, which provided new insights for RB treatment.

Inhibiting \u03b2-catenin disables nucleolar functions in triple-negative breast cancer

Triple-negative breast cancer (TNBC) patients with upregulated Wnt/β-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for β-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear β-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of β-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to β-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of β-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for β-catenin in orchestrating nucleolar activity in TNBCs.

Abstract PS17-39: Inhibition of acetate metabolism enhances host anti-tumor immunity

Abstract Acquired resistance to anti-cancer therapy is an enormous challenge. One of the main factors contributing to therapy resistance is tumor hypoxia. The stress imposed by tumor hypoxia forces cancer cells to adapt in order to survive. These metabolically adapted cancer cells are often more invasive, more malignant, and more drug resistant. As a result, the cancer cells that emerge from hypoxic tumor regions are more likely to cause patient relapse. There is therefore a critical need to understand the mechanisms that promote the survival of cancer cells in stressful tumor microenvironments. We previously showed that the enzyme acetyl-CoA synthetase 2 (ACSS2) supports cancer cell metabolism in hypoxic and nutrient-depleted environments. ACSS2 endows cancer cells with the ability to use acetate as an alternative nutrient source to drive acetyl-CoA biosynthesis during stress and genetic silencing of ACSS2 inhibits human breast tumor growth in xenograft models. Given the important role of acetate metabolism in breast cancer we expanded upon our studies by using immunocompetent hosts and syngeneic mouse tumor models. Our results revealed a previously unknown role of ACSS2 in modulating host anti-tumor immunity. We found that ACSS2 deficient tumors are unable to grow when host immunity is intact. Depletion of host immunity (T cells) using genetic or pharmacological models rescues the growth of ACSS2 deficient tumors. Pharmacological inhibition of ACSS2 in tumors in vivo displayed gene signatures associated immune infiltration and activation within the tumor microenvironment. Moreover, ACSS2 deficient breast cancer cell lines show a marked susceptibility to T cell killing in vitro. Our current research demonstrates a novel role for acetate metabolism in supporting tumor extrinsic modulation of host anti-tumor immunity. Since activation of acetate metabolism via ACSS2 is a near universal hallmark of metabolically stressed cancer cells, targeting acetate metabolism represents an unrealized opportunity with significant upside for improving current therapeutic modalities in breast cancer. Citation Format: Katelyn Miller, Zachary T. Schug. Inhibition of acetate metabolism enhances host anti-tumor immunity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-39.

Circulating extracellular vesicles release oncogenic miR-424 in experimental models and patients with aggressive prostate cancer

Extracellular vesicles (EVs) are relevant means for transferring signals across cells and facilitate propagation of oncogenic stimuli promoting disease evolution and metastatic spread in cancer patients. Here, we investigated the release of miR-424 in circulating small EVs or exosomes from prostate cancer patients and assessed the functional implications in multiple experimental models. We found higher frequency of circulating miR-424 positive EVs in patients with metastatic prostate cancer compared to patients with primary tumors and BPH. Release of miR-424 in small EVs was enhanced in cell lines (LNCaPabl), transgenic mice (Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG) and patient-derived xenograft (PDX) models of aggressive disease. EVs containing miR-424 promoted stem-like traits and tumor-initiating properties in normal prostate epithelial cells while enhanced tumorigenesis in transformed prostate epithelial cells. Intravenous administration of miR-424 positive EVs to mice, mimicking blood circulation, promoted miR-424 transfer and tumor growth in xenograft models. Circulating miR-424 positive EVs from patients with aggressive primary and metastatic tumors induced stem-like features when supplemented to prostate epithelial cells. This study establishes that EVs-mediated transfer of miR-424 across heterogeneous cell populations is an important mechanism of tumor self-sustenance, disease recurrence and progression. These findings might indicate novel approaches for the management and therapy of prostate cancer.

Physalis alkekengi var. franchetii Extracts Exert Antitumor Effects on Non-Small Cell Lung Cancer and Multiple Myeloma by Inhibiting STAT3 Signaling.

BackgroundPhysalis alkekengi var. franchetii is an herb that possesses various ethnopharmacological applications. Herein, our current study focuses on the antitumor effect of a combination of physalins, which are regarded as the most representative secondary metabolites from calyces of Physalis alkekengi var. franchetii.Materials and MethodsWe mainly investigated the antitumor activity of the physalins extracted from Physalis alkekengi var. franchetii on both solid and hematologic cancers. The main cells used in this study were NCI-H1975 and U266 cells. The major assays used were the CCK-8 assay, Western blot analyses, immunofluorescence assay and Annexin V assay, and a xenograft mouse model was used.ResultsThe results showed that physalins exhibited a strong antitumoural effect on both non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells by suppressing constitutive STAT3 activity and further inhibiting the downstream target gene expression induced by STAT3 signaling, which resulted in the enhanced apoptosis of tumor cells. Moreover, physalins significantly reduced tumor growth in xenograft models of lung cancer.ConclusionCollectively, these findings demonstrated that the physalins from Physalis alkekengi var. franchetii may potentially act as cancer preventive or chemotherapeutic agents for NSCLC and MM by inhibiting the STAT3 signaling pathway. The present study served as a promising guide to further explore the precise mechanism of Physalis alkekengi var. franchetii in cancer treatment.