Promote Tumor Cell Growth Research Articles

TRNA-derived small RNA 3′U-tRFValCAC promotes tumour migration and early progression in ovarian cancer

IntroductionDespite recent advances in epithelial ovarian cancer (EOC) management, the highly heterogenous histological/molecular tumour background and patients' treatment response obstructs personalised prognosis and therapeutics. Herein, we have studied the role and clinical utility of the novel subclass of tRNA-derived small RNA fragments emerging via 3′-trailer processing of pre-tRNAs (3′U-tRFs) in EOC. MethodsSK-OV-3 and OVCAR-3 cells were used for in vitro study. Following transfection, cell growth and migration were assessed by CCK8 and wound healing assays, respectively. 3′U-tRFs levels were assessed by reverse transcription quantitative PCR (RT-qPCR), following 3′-end RNA polyadenylation. A screening (OVCAD, n = 100) and institutionally independent validation (TU Munich, n = 103) cohorts were employed for survival analysis using disease progression and patients' death as clinical end-points. Bootstrap analysis was performed for internal validation, and decision curve analysis was used to evaluate clinical benefit on disease prognosis. ResultsFollowing primary clinical assessment, target prediction and gene ontology analyses, the 3′U-tRFValCAC (derived from pre-tRNAValCAC) was highlighted to regulate cell proliferation and adhesion, and to correlate with inferior patients' outcome. 3′U-tRFValCAC transfection of SK-OV-3 and OVCAR-3 cells resulted in significantly increased cell growth and migration, in a dose-dependent manner. Elevated tumour 3′U-tRFValCAC levels were associated with significantly higher risk for early progression and worse survival following first-line platinum-based chemotherapy, independently of patients' clinicopathological data, chemotherapy response, and residual tumour. Interestingly, 3′U-tRFValCAC-fitted multivariate models improved risk stratification and provided superior clinical net benefit in prediction of treatment outcome compared to disease established markers. Conclusions3′U-tRFValCAC promotes tumour cell growth and migration and supports modern risk stratification and prognosis in EOC.

Cancer-associated fibroblasts promote tumor cell growth via miR-493-5p in intrahepatic cholangiocarcinoma.

The association between tumor microenvironment (TME) and cancer-associated fibroblasts (CAFs) in intrahepatic cholangiocarcinoma (ICC) progression is poorly understood. This study aimed to reveal whether specific microRNAs (miRNAs) in extracellular vesicles (EVs) derived from CAFs were involved in ICC progression. Conditioned medium (CM) and EVs in the CM of CAFs and normal fibroblasts (NFs) derived from ICC specimens were used to investigate the effects on tumor cell lines. miRNA microarray assay was used to examine the miRNAs of EVs derived from CAFs and NFs in ICC, and the effects of miR-493-5p on tumor cell lines were examined. Additionally, databases were used to identify miR-493-5p targets, and the relationship between prognosis of ICC patients and cocaine- and amphetamine-regulated transcript propeptide (CARTPT), one of the targets of miR-493-5p, expression in ICC tissues was retrospectively analyzed. Compared with NF-derived CM and EVs, CAF-derived CM and EVs promoted cell lines in proliferation, scratch, migration, and invasion assays. miRNA microarray analysis revealed that miR-493-5p was significantly increased in CAF-derived EVs compared to NF-derived EVs. Tumor cell lines transfected with miR-493-5p were promoted in proliferation and scratch assays. Immunohistochemical staining was performed on 76 ICC specimens; both overall and recurrence-free survival rates were significantly worse in the CARTPT-negative group. Univariate and multivariate analyses showed that low CARTPT expression was an independent poor prognostic factor for overall and recurrence-free survival. Overall, our data suggest that CAFs in the ICC TME suppress CARTPT in tumor cells and promote tumor cells via miR-493-5p in EVs.

Adrenomedullin Secreted by Melanoma Cells Promotes Melanoma Tumor Growth through Angiogenesis and Lymphangiogenesis

Metastatic melanoma is an aggressive tumor and can constitute a real therapeutic challenge despite the significant progress achieved with targeted therapies and immunotherapies, thus highlighting the need for the identification of new therapeutic targets. Adrenomedullin (AM) is a peptide with significant expression in multiple types of tumors and is multifunctional. AM impacts angiogenesis and tumor growth and binds to calcitonin receptor-like receptor/receptor activity-modifying protein 2 or 3 (CLR/RAMP2; CLR/RAMP3). In vitro and in vivo studies were performed to determine the functional role of AM in melanoma growth and tumor-associated angiogenesis and lymphangiogenesis. In this study, AM and AM receptors were immunohistochemically localized in the tumoral compartment of melanoma tissue, suggesting that the AM system plays a role in melanoma growth. We used A375, SK-MEL-28, and MeWo cells, for which we demonstrate an expression of AM and its receptors; hypoxia induces the expression of AM in melanoma cells. The proliferation of A375 and SK-MEL-28 cells is decreased by anti-AM antibody (αAM) and anti-AMR antibodies (αAMR), supporting the fact that AM may function as a potent autocrine/paracrine growth factor for melanoma cells. Furthermore, migration and invasion of melanoma cells increased after treatment with AM and decreased after treatment with αAMR, thus indicating that melanoma cells are regulated by AM. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing melanoma cells, as demonstrated by reduced numbers of vessel structures, which suggests that AM is one of the melanoma cells-derived factors responsible for endothelial cell-like and pericyte recruitment in the construction of neovascularization. In vivo, αAMR therapy blocked angiogenesis and lymphangiogenesis and decreased proliferation in MeWo xenografts, thereby resulting in tumor regression. Histological examination of αAMR-treated tumors showed evidence of the disruption of tumor vascularity, with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. The expression of AM by melanoma cells promotes tumor growth and neovascularization by supplying/amplifying signals for neoangiogenesis and lymphangiogenesis.

Long Noncoding RNA LINC01410 Interacts with the Minichromosome Maintenance (MCM) Complex to Promote Tumor Cell Growth in Multiple Myeloma

The human genome contains thousands of loci that produce long non-protein coding RNAs (lncRNAs) that play critical roles in healthy cell function as well as tumorigenesis. We have recently described the lncRNA landscape in newly diagnosed multiple myeloma (MM), identified their role as independent risk predictors for clinical outcome, and validated the functional contribution of specific lncRNAs to MM cell growth and viability. Although these examples suggest the significance of lncRNAs in MM pathobiology, the vast majority of lncRNA genes have not been functionally tested. To identify lncRNAs that promote MM cell growth, we performed a large-scale cell viability screen of lncRNAs using CRISPR interference (CRISPRi) to inhibit gene expression. To find lncRNA targets, we first interrogated our large RNA-seq dataset from 360 newly diagnosed MM patients and identified 913 lncRNA transcripts expressed in primary MM cells. We designed the CRISPRi library to target the transcription start site of these 913 lncRNA genes, each with 7 sgRNAs. We screened 3 MM cell lines (H929, KMS12BM, KMS11) and identified 32 lncRNA loci that modified cell growth upon CRISPRi targeting, with 7 lncRNAs impacting all three cell lines tested. We then evaluated the differential expression of these lncRNAs in primary MM cells compared to normal plasma cells (PC). We observed that LINC01410 was significantly overexpressed in MM patients compared to normal PC, especially in MM patients with t(4;14) translocations, and was an independent risk predictor that highly correlated with overall survival in newly diagnosed MM patients. We next knocked down LINC01410 using gapmeR antisense oligonucleotides (ASO) in a panel of MM cell lines and observed time-dependent inhibition of MM cell viability, including in the t(4;14) cell lines. As LINC01410 appears to localize to both the cytosol and nucleus, we also utilized siRNAs to specifically target the cytosolic isoform and observed a robust reduction of cell growth, suggesting that both cellular localizations are important for the activity of LINC01410. Transcriptomic analysis indicated a set of genes modulated in response to LINC01410 depletion, with an enrichment in genes involved in the cell cycle and DNA replication. An RNA-protein pull down (RPPD) assay identified three out of six MCM complex subunits (MCM4, MCM5, MCM7) as relevant protein interactors of LINC01410 and was confirmed using RNA-immunoprecipitation (RIP) assay. The MCM complex is a DNA helicase essential for genomic DNA replication, and we hypothesize that LINC01410 affects cell proliferation by interacting with this complex. We are now investigating this functional interaction by looking at the impact of LINC01410 modulation on the assembly of the complex, by using integrated mass spectrometry analysis, and on its ability to load onto chromatin by using chromatin immunoprecipitation sequencing (ChIP-Seq). In conclusion, we report the identification of LINC01410 as a novel lncRNA dependancy that supports MM cell growth via interacting with the MCM complex and controlling the cell cycle/DNA replication axis. Promising developments in the field of lncRNA inhibitors may allow forthcoming clinical applications.

PHF19 Inhibits Multiple Myeloma Cell Response to Immunotherapy Via Promoting Immunosuppressive Microenvironment

Introduction: The multiple myeloma (MM) bone marrow (BM) microenvironment promotes tumor cell growth, survival, and drug resistance, while also conferring immunosuppression. This involves crosstalk between the myeloma cells and the components of BM microenvironment which can be facilitated or hindered by epigenetic modifiers altering the gene expression of receptors. Among those, regulatory T (Tregs) cells are essential to control peripheral tolerance and response to foreign and tumor antigens. Tregs within the BM niche play a significant role in the suppression of antitumor immune responses and limiting the beneficial response of immunotherapy in MM. Here, we investigated the impact of the Polycomb-like protein PHF19/PCL3, an epigenetic gene whose high level predicts poor prognosis in patients with MM, on the immunosuppressive BM microenvironment. Methods & Results: We first investigated how PHF19 overexpression would affect the microenvironment by using an ex vivo co-culture of MM cells with PBMCs or purified CD3+ T cells. MM cells that overexpressed PHF19 by lenti-virus infection (PHF19-OE cells) induced 2-4-fold higher conversion of conventional T cells (Tcon) into induced regulatory T cells (iTregs) (n=5, day 4). In addition, PHF19-OE MM cells increased the frequency of iTreg at later time points (day 7) and upregulated cell proliferation (CCND1, CCND2), anti-apoptosis (BCL2, BCL2L1), and Treg-related genes (TGFβ1, IL-10, Foxp3) (P < .0001). Moreover, enhanced checkpoint marker (PD1, TIM3, LAG3) expression was observed on Tcon co-cultured with PHF19-OE vs control MM cells. There were elevated IL-10 and TGFβ1 levels in the culture supernatants of PHF19-OE MM cells, alone and in co-cultures, and antibodies to these cytokines partially neutralized the Treg inhibition of Tcon proliferation triggered by PHF19-OE MM cells. Thus, PHF19 seems to be important in facilitating the crosstalk between MM cells and immune cells in the BM. CD38 expression on the MM cell surface is commonly targeted in immunotherapy. However, CD38 expression was downregulated on PHF19-OE vs -KD MM cells, and PHF19 mRNA expression was negatively correlated to CD38 expression in patient MM cells (Spearman's ρ = -0.38 and -0.2 for GSE5900 and GSE31161, P < .004). As expected, PHF19-OE MM cells were less susceptible than PHF19-KD MM cells to in vitro NK-mediated lysis induced by daratumumab (dara) or isatuximab, which target CD38, as evidenced by fewer CD107a+ and IFN γ+ NK effector cells. Moreover, dara-induced cytotoxicity was diminished in xenografted NSG mice with PHF19-OE vs control cells and reconstituted with human NK cells. These resistant PHF19-OE AMO1 tumors had lower expression of CD38, STAT1, and IFN-related genes, which produced higher levels of human κ light chain, soluble BCMA, and TGFβ1 in matched serum samples. Accordingly, PHF19-OE resulted in significantly decreased mouse survival. To find the mediators of the immunosuppression induced by PHF19, we used ATAC-seq and CHIP-seq analysis in MM cell lines. We found that a number of genes essential for T cell-mediated tumor immunity was downregulated in the PHF19 OE group vs control, including key genes in the interferon-γ (IFN-γ) signaling pathway, which is a pathway that leads to cytokine-mediated cell death. Among the genes of the IFN-γ pathway, 56 genes had less chromatin accessibility in PHF19 OE vs control cells. RNA-seq analysis confirmed that PHF19 OE repressed the expression of CD38 and the IFN-γ and IFN-α signaling pathway. Furthermore, PHF19 OE altered the expression of genes in NF-κB signaling triggered by TNF-α signaling pathway, which are associated with resistance to immunotherapy. Finally, activation of the PI3K/AKT pathway was also identified in PHF19 OE cells, which directly promotes MM cell proliferation and survival, as well as enhances resistance to immunotherapy via upregulation of PD-L1 on the MM cell surface. Conclusions: Taken together, these data indicate that PHF19 promotes an immunosuppressive BM milieu by promoting iTreg frequency and the expression of immunosuppressive cytokines, as well as decreasing CD38 expression on MM cells and diminishing their sensitivity to IFN-γ-mediated killing. These results suggest the potential clinical utility of targeting PHF19 to overcome immunosuppression and thereby enhance or restore sensitivity to CD38-targeted therapy in MM.

NEK2 is associated with poor prognosis of clear cell renal cell carcinoma and promotes tumor cell growth and metastasis

BackgroundNever in Mitosis gene-A(NIMA)-related Kinase 2 (NEK2) is a critical player in themitotic processes. NEK2 is highly expressed in many kindsof human cancers and has been shown toparticipatein drug resistance, tumorigenesis, and tumor progression. However, the expression or function of NEK2 in clear cell renal cell carcinoma (ccRCC)hasnot yet been investigated. MethodsWeused TCGA databaseto study the NEK2 expression in ccRCC. The expression of NEK2 in tumor tissuesand adjacent tissueswas examined by immunohistochemistry. We also analysed the correlation between NEK2 expression and clinical parametersofccRCC. The mRNA and protein level of NEK2 expression were semi-quantifiedby qRT-PCR and western blotting analysis. Following NEK2 knockdown by RNA interference in Caki-1cells, whileNEK2 overexpression in A489 cells, CCK8and transwell assay was used to confirmtheproliferation, migration and invasion, respectively.Finally, our in vivo study were carried out using nudemice to establish mouse model for kidney cancer. ResultsWe observed elevated expression of NEK2 both in ccRCCtumor tissues and cell lines. Together with clinical and pathological features, our analysis indicated a clear association of clinical outcomes between ccRCC patients with high and lowNEK2expression. Our in vitro studies demonstratedthat NEK2 knockdowninhibits the proliferation,migrationand invasion of Caki-1cells, oppositely, overexpressionof NEK2 promotes the proliferation, migrationand invasion of A489cells.In the end, our animal study demonstrated that deletion of NEK2 expression could impair tumor growth. ConclusionOur data suggestedthat NEK2wasimportant inregulating ccRCC cell proliferation and metastasis, and indicated NEK2as a potentially important target for the treatment ofccRCC.

Chromosome 12 Open Reading Frame 49 Promotes Tumor Growth and Predicts Poor Prognosis in Colorectal Cancer

Background and AimsLittle is known about the role of chromosome 12 open reading frame 49 (C12ORF49)-induced metabolic signal transduction in tumor growth. We investigated the relationship between C12ORF49 expression and prognosis in colorectal cancer (CRC) patients.MethodsC12ORF49 protein expression was measured in CRC tissues by Western blot and immunohistochemistry staining. Knock out of C12ORF49 in CRC cells was then performed, and the role of C12ORF49 in CRC cell proliferation and growth was examined. The expression of C12ORF49 in CRC was analyzed in Gene Expression Profiling Interactive Analysis (GEPIA) databases. A prognosis model with 11 C12ORF49-associated genes (CAGs) was generated by TCGA databases.ResultsC12ORF49 expression was significantly higher in CRC tumor tissue than in non-tumor tissue. Furthermore, in vitro and in vivo loss-of-function experiments, showed that C12ORF49 plays critical roles in promoting tumor cell growth. There was a significant correlation between C12ORF49 protein and the presence of tumor necrosis. C12ORF49 is critical for its interaction with SREBF1, TMEM41A, and S1PR3 in the poor prognosis of CRC.ConclusionsOur results suggest that C12ORF49 plays a key role in CRC tumor growth.

HER2 Overexpression and Cytogenetical Patterns in Canine Mammary Carcinomas.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical-pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical-pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

TAOK1 Promotes Proliferation and Invasion of Non-Small-Cell Lung Cancer Cells by Inhibition of WWC1.

Background For patients with advanced non-small-cell lung cancer (NSCLC), targeted therapy significantly improves the therapeutic effect of NSCLC patients. With the development of molecular targeted therapy, more and more NSCLC-related genes have been found. Thousand and one amino-acid kinase 1 (TAOK1) has been identified as a potential target for drug research in various cancers. The main objective of this study was to explore the expression and function of TAOK1 in NSCLC. Methods Western blotting was employed to assess TAOK1 expression in NSCLC cell lines. The effects of TAOK1 on biological behaviors, including proliferation, invasion, and apoptosis of NSCLC cells, were assessed. The relationship between TAOK1 and WW and C2 domain containing 1 (WWC1) was assessed by Co-IP assay. The subcutaneous injection of tumor cells in nude mice was used to verify it in vivo. Results As expected, TAOK1 was increased in NSCLC cell lines. Following TAOK1 knockdown, NSCLC cells exhibited a significant decrease in the invasion and increased apoptosis in vitro. Instead, the TAOK1 elevation showed the opposite results. The Co-IP assay identified that TAOK1 specifically interacted with WWC1. Knockdown of WWC1 overturned TAOK1 silencing-mediated malignant phenotype of NSCLC cells. Additionally, subcutaneous tumorigenesis assays in nude mice confirmed that TAOK1 knockdown markedly restrained the proliferation capacity of NSCLC cells in vivo. Conclusion Surprisingly, TAOK1 overexpression in NSCLC promotes tumor cell growth and invasion, which is associated with downregulation of its downstream protein WWC1, and this result might provide a robust research basis to inquire about the precise therapeutic targets for NSCLC.

Tumor-associated macrophages promote migration and invasion via modulating IL-6/STAT3 signaling in renal cell carcinoma

Tumor-associated macrophages (TAMs) promote tumor cell growth and metastasis in various human cancers. However, the role of TAMs in renal cell carcinoma (RCC) is rarely investigated. Herein, we observed that the infiltration of TAMs was obviously elevated in RCC tumor tissues, high infiltration of TAMs was closely associated with tumor progression and poor prognosis in RCC patients. In vitro assays further indicated that the conditioned medium of TAMs (TAMs CM) facilitated migration, invasion, and epithelial-mesenchymal transition (EMT) in RCC cells. Moreover, we found that IL-6 was involved in the functions of TAMs in RCC; IL-6 neutralizing antibody (IL-6NA) partly abolished the effect of TAMs on RCC cells. In addition, we demonstrated that TAMs might exert their roles by activating STAT3 signaling in RCC, and IL-6 was responsible for TAMs-induced STAT3 signaling activation. In conclusion, our results revealed that high infiltration of TAMs may promote RCC cells migration, invasion, and EMT via modulating IL-6/STAT3 signaling, further suggesting a potential of novel treatment strategies targeting TAMs or IL-6 for metastatic RCC.

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

SummaryIon and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K+ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K+ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.

P-101: Long noncoding RNA LINC01410 interacts with the minichromosome maintenance (MCM) complex to promote tumor cell growth in multiple myeloma

P-101: Long noncoding RNA LINC01410 interacts with the minichromosome maintenance (MCM) complex to promote tumor cell growth in multiple myeloma

The AMPK-HOXB9-KRAS axis regulates lung adenocarcinoma growth in response to cellular energy alterations.

HOXB9 is an important transcription factor associated with unfavorable outcomes in patients with lung adenocarcinoma (LUAD). However, its degradation mechanism remains unclear. Here, we show that HOXB9 is a substrate of AMP kinase alpha (AMPKα). AMPK mediates HOXB9 T133 phosphorylation and downregulates the level of HOXB9 in mice and LUAD cells. Mechanistically, phosphorylated HOXB9 promoted E3 ligase Praja2-mediated HOXB9 degradation. Blocking HOXB9 phosphorylation by depleting AMPKα1/2 or employing the HOXB9 T133A mutant promoted tumor cell growth in cell culture and mouse xenografts via upregulation of HOXB9 and KRAS that is herein identified as a target of HOXB9. Clinically, AMPK activation levels in LUAD samples were positively correlated with pHOXB9 levels; higher pHOXB9 levels were associated with better survival of patients with LUAD. We thus present a HOXB9 degradation mechanism and demonstrate an AMPK-HOXB9-KRAS axis linking glucose-level-regulated AMPK activation to HOXB9 stability and KRAS gene expression, ultimately controlling LUAD progression.

Regulation and Function of Matrix Metalloproteinase-13 in Cancer Progression and Metastasis.

Simple SummaryMMP-13 is an enzyme that can digest and disrupt the tissue structures surrounding epithelial cells that line the surface of many internal organs, as well as the tissue structures surrounding endothelial cells that line the surface of blood vessels. The production of MMP-13 is tightly controlled in physiological conditions but is increased in various cancers and plays multiple roles in tumour progression and metastasis. This review summarises the current understanding of the regulation of MMP-13 production and discusses the actions of MMP-13 in cancer progression and metastasis.Matrix metalloproteinase-13 (MMP-13) is a member of the Matrix metalloproteinases (MMPs) family of endopeptidases. MMP-13 is produced in low amounts and is well-regulated during normal physiological conditions. Its expression and secretion are, however, increased in various cancers, where it plays multiple roles in tumour progression and metastasis. As an interstitial collagenase, MMP-13 can proteolytically cleave not only collagens I, II and III, but also a range of extracellular matrix proteins (ECMs). Its action causes ECM remodelling and often leads to the release of various sequestered growth and angiogenetic factors that promote tumour cell growth, invasion and angiogenesis. This review summarizes our current understanding of the regulation of MMP-13 expression and secretion and discusses the actions of MMP-13 in cancer progression and metastasis.

BARX2/FOXA1/HK2 axis promotes lung adenocarcinoma progression and energy metabolism reprogramming.

BackgroundMetabolic reprogramming is an emerging cancer feature that has recently drawn special attention since it promotes tumor cell growth and proliferation. However, the mechanism of the Warburg effect is still largely unknown. This research aimed to reveal the effects of BarH-like homeobox 2 (BARX2) in regulating tumor progression and glucose metabolism in lung adenocarcinoma (LUAD).MethodsExpression of BARX2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in LUAD cell line and tissues, and the tumor-promoting function of BARX2 in LUAD cells was detected in vitro and in vivo xenograft models. The metabolic effects of BARX2 were examined by detecting glucose uptake, the production levels of lactate and pyruvate, and the extracellular acidification rate (ECAR). Chromatin immunoprecipitation (ChIP) assay and luciferase reporter gene assay were used to identify the underlying molecular mechanism of BARX2 regulation of HK2. Further studies showed that transcription factor FOXA1 directly interacts with BARX2 and promotes the transcriptional activity of BARX2.ResultsBARX2 was remarkably up-regulated in LUAD tissues and positively linked to advanced clinical stage and poor prognosis. In vitro and in vivo data indicated ectopic expression of BARX2 enhanced cell proliferation and tumorigenesis, whereas BARX2 knockdown suppressed these effects. Metabolic-related experiments showed BARX2 promoted the reprogramming of glucose metabolism. Mechanistically, the BARX2/FOXA1/HK2 axis promoted LUAD progression and energy metabolism reprogramming.ConclusionsIn summary, our research first defined BARX2 as a tumor-promoting factor in LUAD and that it may act as a novel prognostic biomarker and new therapeutic target for the disease.

ILP-2: A New Bane and Therapeutic Target for Human Cancers

Inhibitor of apoptosis protein-related-like protein-2 (ILP-2), also known as BIRC-8, is a member of the inhibitor of apoptosis protein (IAPs) family, which mainly encodes the negative regulator of apoptosis. It is selectively overexpressed in a variety of human tumors and can help tumor cells evade apoptosis, promote tumor cell growth, increase tumor cell aggressiveness, and appears to be involved in tumor cell resistance to chemotherapeutic drugs. Several studies have shown that downregulation of ILP-2 expression increases apoptosis, inhibits metastasis, reduces cell growth potential, and sensitizes tumor cells to chemotherapeutic drugs. In addition, ILP-2 inhibits apoptosis in a unique manner; it does not directly inhibit the activity of caspases but induces apoptosis by cooperating with other apoptosis-related proteins. Here, we review the current understanding of the various roles of ILP-2 in the apoptotic cascade and explore the use of interfering ILP-2, and the combination of related anti-tumor agents, as a novel strategy for cancer therapy.

Abstract 503: Longitudinal minimally invasive monitoring of resistance mutations in ALK rearranged lung cancer patients

Abstract Background: ALK is a proto-oncogene encoding the receptor tyrosine kinase ALK, which can be aberrantly activated by gene rearrangements or point mutations to promote tumor cell growth, survival, and metastasis. ALK rearrangements are detected in approximately 4% of patients with advanced non-small cell lung cancer (NSCLC), and the tyrosine kinase inhibitors (TKIs) crizotinib, alectinib, brigatinib, ceritinib, and lorlatinib have been approved by the FDA for the treatment of ALK-positive NSCLC. The durability of response to these therapies has been limited, in many cases, by the emergence of mutations that confer resistance to ALK. The major resistance mutation to alectinib, brigatinib, and ceritinib is the ALK G1202R solvent front mutation. The detection of resistance mutations has been improved with the evolution of biotechnology, making it possible to search for tumor-derived plasma free DNA as a liquid biopsy in cases where tissue biopsies are difficult to obtain. Digital PCR (dPCR) is an improved method of the conventional PCR method, which is more accurate and allows absolute quantification than the RT-PCR method. Materials and Methods: For a total of 9 patients, 2 patients newly diagnosed with ALK+ NSCLC by tissue biopsy and about to receive ALK-TKI and 7 patients already diagnosed with ALK+ NSCLC and receiving ALK-TKI, 10 ml of plasma was collected in addition to the blood collection at each 3-monthly visit. cfDNA was extracted and digital PCR was used to simultaneously detect 10 ALK mutations (T1151ins, C1156Y, L1196M, G1269A, F1174L, L1152R, V1180L, I1171T, G1202R, S1206Y). This Study was supported by an Investigator Initiated Research (IIR) from Pfizer. Results: The total number of samples analyzed in 9 cases was 65, and the median number of samples collected in each case was 7 (minimum 2 - maximum 12). cfDNA concentration was a median of 9.9 ng/ml of plasma (4.0-35.8 ng/ml). The number of samples detected for each mutation was 32 for G1202R, 16 for C1156Y, 5 for G1269A, 4 for F1174L, 1 for T1151ins, and 1 for I1171T. G1202R was detected in 19 of 37 samples during treatment with alectinib. The median time from detection of G1202R to clinical disease progression was 388 days (123-2004 days), with repeated emergence and disappearance in some cases. Conclusions: The dPCR method was highly sensitive in detecting gene mutations with low allele frequency. It was also found to be able to monitor variable resistance mutations over time during ALK-TKI treatment. In addition, we found that the appearance of G1202R did not always indicate clinical acquisition of resistance that required drug modification. A limitation of this study is that the mutations detected are not distinguishable between the same allele or Cis/trans. In particular, EML4-ALK forms a trimeric protein, suggesting that single mutations such as G1202R and drug susceptibility do not always match in clinical specimens. Citation Format: Takaaki Sasaki, Noriko Hirai, Yoshinori Minami, Shunsuke Okumura, Shin-ichi Chiba, Ryohei Yoshida. Longitudinal minimally invasive monitoring of resistance mutations in ALK rearranged lung cancer patients [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 503.

Abstract 6383: Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis

Abstract Cancer stem-like cells (CSCs) and the differentiated bulk cells mainly contribute to cellular heterogeneity within a tumor. The dichotomous division pattern of the CSCs sitting atop the tumor hierarchy allow them to self-renew, and differentiate to initiate and repopulate aggressive tumors. These CSC-differentiation-derived bulk tumor cells reprogram to stem-like states, accounting for high plasticity within a tumor; a phenomenon governed by the distinct epigenetic profiles of the two cell populations. Reciprocal signaling between the tumor and its complex microenvironment is a vital driver of plasticity and heterogeneity in cancer. The indispensable role of CSCs in cellular cross-talk in the tumor niche as critical drivers of tumorigenesis is well-studied. However, the massive outnumbering of the CSCs by the differentiated cells raises several vital questions regarding the role of the latter in tumor growth; whether they contribute to the complex signaling within the tumor microenvironment (TME) to drive tumor growth remains unexplored. In this study, we aimed to mechanistically delineate the distinct roles of the cancer stem-like and differentiated cells by investigating their interactions with other stromal cells in the TME.In our model system, Glioblastoma (GBM), a highly aggressive brain cancer, we adopted a quantitative, label-free, high-throughput proteomics-based approach to identify the differentially abundant secreted proteins between the glioma stem-like cell (GSC) and differentiated glioma cell (DGC). This analysis showed a significantly higher abundance of the Fibromodulin (FMOD), a collagen-binding proteoglycan, in the conditioned medium of the DGCs compared with that of the GSCs. Our investigation revealed that an activated TGF-β signaling leads to p-SMAD2 occupancy on the FMOD promoter region, leading to increased FMOD secretion by the DGCs. DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth in a subcutaneous mouse model. We also observed that FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. Moreover, DGC-secreted FMOD activates integrin-dependent Notch signaling in endothelial cells, promoting angiogenesis. FMOD-silencing also inhibits the capability of the transdifferentiated endothelial cells (derived from the DGCs) to form angiogenic networks. Conditional silencing of FMOD in the de novo formed DGCs in an orthotopic intracranial C57BL/6 mouse model inhibited the growth of GSC-initiated tumors due to poorly developed vasculature, thereby increasing mouse survival. The same observation was recapitulated using patient-derived GSCs in an immunocompromised intracranial mouse model, thereby highlighting the human relevance of our findings. Collectively, our findings demonstrate how paracrine signaling between DGC-secreted FMOD and the stromal cells promotes tumor growth by enhancing angiogenesis, thereby highlighting a previously less studied supportive role of DGCs in tumor growth, albeit the GSCs retaining the tumor-initiating capacity. Thus our study identifies DGC-specific FMOD as a potential therapeutic target and emphasizes that targeting both cancer stem cells and differentiated cancer bulk cells is vital to achieving a durable response in GBM. Citation Format: Shreoshi Sengupta, Kumaravel Somasundaram, Dinorah Friedmann Morvinski, Philippe Marin, Prasasvi Reddy, Arani Mukherjee, Prerna Magod, Mainak Mondal, Serge Urbach. Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis [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 6383.

The glycocalyx in tumor progression and metastasis

Tumors develop a desmoplastic response that creates a tissue microenvironment that is complicit in malignant transformation and metastasis. Tumor desmoplasia is characterized by fibrosis that is mediated by elevated extracellular matrix deposition, remodeling and cross‐linking that stiffen the tumor stroma. We have been studying how these aberrant cell and tissue level forces promote malignant transformation and drive tumor metastasis, and how they modulate tumor recurrence and treatment resistance in breast and pancreatic cancer and glioblastoma. We use two and three dimensional culture models with tuned extracellular matrix stiffness, as well as transgenic and syngeneic mouse models, human PDX models and human biospecimens, in which ECM crosslinking and stiffness and integrin mechanosignaling can be quantified and modified. Our studies have thus far revealed that the ECM in all tumors becomes progressively remodeled and stiffened by the resident stromal fibroblasts and revealed that this occurs prior to malignant transformation. The stromal‐fibroblast stiffened ECM disrupts tissue organization, promotes tumor cell growth, survival and invasion, induces angiogenesis, and promotes a pro‐tumor immunity that drives tumor aggression and metastasis. We recently found that the fibrotic stroma drives the metabolic rewiring of the tumor cells and determined that this associates with elevated expression of key enzymes that regulate the cellular glycoproteome. A stiffened fibrotic stroma stimulated sialoglycan expression through tension‐induced integrin signaling. The elevated mechanosignaling induced in the tumor cells by the stiffened stroma increased the level of secreted proteoglycans deposited at the tumor‐ECM interface where a high density of protumorigenic immune suppressive infiltrating myeloid cells were detected. The association between a stiff, fibrotic tumor stroma, elevated secreted proteoglycans and a pro tumor immune infiltrate was very predominant in the more aggressive human breast tumors. I will discuss the dynamic and reciprocal interplay between tissue tension, metabolic reprogramming and glycoprotein regulation and innate and acquired immunity in tumor aggression and progression.

PI3K Inhibitors in Advanced Breast Cancer: The Past, The Present, New Challenges and Future Perspectives.

Simple SummaryMolecular aberrations in the phosphatidylinositol 3-kinase (PI3K) pathway are often observed in breast cancer and represent a key regulator of many cellular processes, promoting tumor cell growth and survival. The first clinical trials leading to the development of pan-PI3K inhibitors showed certain preclinical activity; nevertheless, the toxicity profile limited further analysis of this drugs’ class. To improve the antitumor effect and therapeutic index, additional clinical trials have been performed to develop the PI3K isoform-specific inhibitors and new schedule combinations with a good toxicity profile. However, further efforts are needed to discover other potentially actionable genetic alterations that remain a challenge to reach the goal of personalized and precision medicine.Breast cancer is the leading cause of death in the female population and despite significant efforts made in diagnostic approaches and treatment strategies adopted for advanced breast cancer, the disease still remains incurable. Therefore, development of more effective systemic treatments constitutes a crucial need. Recently, several clinical trials were performed to find innovative predictive biomarkers and to improve the outcome of metastatic breast cancer through innovative therapeutic algorithms. In the pathogenesis of breast cancer, the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/AKT)-mammalian target of rapamycin (mTOR) axis is a key regulator of cell proliferation, growth, survival, metabolism, and motility, making it an interest and therapeutic target. Nevertheless, the PI3K/AKT/mTOR cascade includes a complex network of biological events, needing more sophisticated approaches for their use in cancer treatment. In this review, we described the rationale for targeting the PI3K pathway, the development of PI3K inhibitors and the future treatment directions of different breast cancer subtypes in the metastatic setting.