IQGAP3 signalling mediates intratumoral functional heterogeneity to enhance malignant growth.

BACKGROUND: The elevation of IQGAP3 expression in diverse cancers indicates a key role for IQGAP3 in carcinogenesis. Although IQGAP3 was established as a proliferating stomach stem cell factor and a regulator of the RAS-ERK pathway, how it drives cancer growth remains unclear. OBJECTIVE: We define the function of IQGAP3 in gastric cancer (GC) development and progression. DESIGN: We studied the phenotypic changes caused by IQGAP3 knockdown in three molecularly diverse GC cell lines by RNA-sequencing. In vivo tumorigenesis and lung metastasis assays corroborated IQGAP3 as a mediator of oncogenic signalling. Spatial analysis was performed to evaluate the intratumoral transcriptional and functional differences between control tumours and IQGAP3 knockdown tumours. RESULTS: Transcriptomic profiling showed that IQGAP3 inhibition attenuates signal transduction networks, such as KRAS signalling, via phosphorylation blockade. IQGAP3 knockdown was associated with significant inhibition of MEK/ERK signalling-associated growth factors, including TGFß1, concomitant with gene signatures predictive of impaired tumour microenvironment formation and reduced metastatic potential. Xenografts involving IQGAP3 knockdown cells showed attenuated tumorigenesis and lung metastasis in immunodeficient mice. Accordingly, immunofluorescence staining revealed significant reductions of TGFß/SMAD signalling and αSMA-positive stromal cells; digital spatial analysis indicated that IQGAP3 is indispensable for the formation of two phenotypically diverse cell subpopulations, which played crucial but distinct roles in promoting oncogenic functions. CONCLUSION: IQGAP3 knockdown suppressed the RAS-TGFß signalling crosstalk, leading to a significant reduction of the tumour microenvironment. In particular, IQGAP3 maintains functional heterogeneity of cancer cells to enhance malignant growth. IQGAP3 is thus a highly relevant therapy target in GC.

Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer.

Gastric cancer heterogeneity represents a barrier to disease management. We generated a comprehensive single-cell atlas of gastric cancer (>200,000 cells) comprising 48 samples from 31 patients across clinical stages and histologic subtypes. We identified 34 distinct cell-lineage states including novel rare cell populations. Many lineage states exhibited distinct cancer-associated expression profiles, individually contributing to a combined tumor-wide molecular collage. We observed increased plasma cell proportions in diffuse-type tumors associated with epithelial-resident KLF2 and stage-wise accrual of cancer-associated fibroblast subpopulations marked by high INHBA and FAP coexpression. Single-cell comparisons between patient-derived organoids (PDO) and primary tumors highlighted inter- and intralineage similarities and differences, demarcating molecular boundaries of PDOs as experimental models. We complemented these findings by spatial transcriptomics, orthogonal validation in independent bulk RNA-sequencing cohorts, and functional demonstration using in vitro and in vivo models. Our results provide a high-resolution molecular resource of intra- and interpatient lineage states across distinct gastric cancer subtypes. SIGNIFICANCE: We profiled gastric malignancies at single-cell resolution and identified increased plasma cell proportions as a novel feature of diffuse-type tumors. We also uncovered distinct cancer-associated fibroblast subtypes with INHBA-FAP-high cell populations as predictors of poor clinical prognosis. Our findings highlight potential origins of deregulated cell states in the gastric tumor ecosystem. This article is highlighted in the In This Issue feature, p. 587.

Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids.

(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

Hedgehog transcriptional effector GLI mediates mTOR-Induced PD-L1 expression in gastric cancer organoids.

Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient's tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.

DNA epigenetic signature predictive of benefit from neoadjuvant chemotherapy in oesophageal adenocarcinoma: results from the MRC OE02 trial.

BACKGROUND: DNA methylation signatures describing distinct histological subtypes of oesophageal cancer have been reported. We studied DNA methylation in samples from the MRC OE02 phase III trial, which randomised patients with resectable oesophageal cancer to surgery alone (S) or neoadjuvant chemotherapy followed by surgery (CS). AIM: The aim of the study was to identify epigenetic signatures predictive of chemotherapy benefit in patients with oesophageal adenocarcinoma (OAC) from the OE02 trial and validate the findings in an independent cohort. METHODS: DNA methylation was analysed using the Illumina GoldenGate platform on surgically resected OAC specimens from patients in the OE02 trial. Cox proportional hazard analysis was performed to select probes predictive of survival in the CS arm. Non-negative matrix factorisation was used to perform clustering and delineate DNA methylation signatures. The findings were validated in an independent cohort of patients with gastroesophageal adenocarcinoma treated with neoadjuvant chemotherapy. RESULTS: A total of 229 patients with OAC were analysed from the OE02 trial (118 in the CS arm and 111 in the S arm). There was no difference in DNA methylation status between the CS and S arms. A metagene signature was created by dichotomising samples into two clusters. In cluster 1, patients in the CS arm had significant overall survival (OS) benefit (median OS CS: 931 days vs. S: 536 days [HR: 1.54, P = 0.031]). In cluster 2, patients in the CS arm had similar (or worse) OS compared with patients in the S arm (CS: 348 days vs. S: 472 days [HR: 0.70, P = 0.1], and test of interaction was significant (p = 0.005). In the validation cohort (n = 13), there was no difference in DNA methylation status in paired pre- and post-treatment samples. When the epigenetic signature was applied, cluster 1 samples had better OS (median OS, cluster 1: 1174 days vs. cluster 2: 392 days, HR: 3.47, p = 0.059) CONCLUSIONS: This is the first and largest study of DNA methylation in patients with OAC uniformly treated in a randomised phase III trial. We identified an epigenetic signature that may serve as a predictive biomarker for chemotherapy benefit in OAC.

Phospho­STAT1 expression as a potential biomarker for anti­PD­1/anti­PD­L1 immunotherapy for breast cancer.

In the present study, we evaluated the mechanisms of programmed death ligand 1 (PD­L1) expression in the breast cancer microenvironment, focusing on the role of interferon­Î³ (IFN­Î³), and the clinical indications for anti­programmed cell death 1 (PD­1) /anti­PD­L1 immunotherapy. We evaluated PD­L1 expression in 4 breast cancer cell lines in the presence of 3 types of inhibitors, as well as IFN­Î³. The expression of phosphorylated signal transducer and activator of transcription 1 (p­STAT1), one of the IFN­Î³ signaling pathway molecules, was analyzed using immunohistochemistry (IHC) in relation to PD­L1 and human leukocyte antigen (HLA) class I expression on cancer cells and tumor­infiltrating CD8­positive T cells in 111 patients with stage II/III breast cancer. Using The Cancer Genome Atlas (TCGA) database, the correlation of the IFN­Î³ signature with PD­L1 expression was analyzed in breast invasive carcinoma tissues. As a result, the JAK/STAT pathway via IFN­Î³ was mainly involved in PD­L1 expression in the cell lines examined. IHC analysis revealed that the PD­L1 and HLA class I expression levels were significantly upregulated in the p­STAT1­positive cases. TCGA analysis indicated that the PD­L1 expression and IFN­Î³ signature exhibited a positive correlation. On the whole, these findings suggest that PD­L1 and HLA class I are co­expressed in p­STAT1­positive breast cancer cells induced by IFN­Î³ secreted from tumor infiltrating immune cells, and that p­STAT1 expression may be a potential biomarker for patient selection for immunotherapy with anti­PD­1/anti­PD­L1 monoclonal antibodies.

Real-Time Tumor Gene Expression Profiling to Direct Gastric Cancer Chemotherapy: Proof-of-Concept "3G" Trial.

Purpose: The oxaliplatin plus S-1 and cisplatin plus S-1 regimens are interchangeably used in the management of advanced gastric cancer. The previously reported G-intestinal (G1) and G-diffuse (G2) intrinsic gene expression signatures showed promise for stratifying patients according to their tumor sensitivity to oxaliplatin or cisplatin.Experimental Design: The proof-of-concept, multicenter, open-label phase II "3G" trial was done to prospectively evaluate the feasibility and efficacy of using genomic classifiers to tailor treatment in gastric cancer. Patients' tumors were classified as "G1" or "G2" using a nearest-prediction template method, or "G3" (unclear assignment) when FDR ≥ 0.05. The first 30 patients in the "G1" cohort were assigned oxaliplatin plus S-1 (SOX) chemotherapy; thereafter, subsequently recruited "G1" patients were treated with cisplatin plus S-1 (SP) chemotherapy. "G2" patients and "G3" patients were treated with SP and SOX chemotherapy, respectively.Results: A total of 48, 21, and 12 patients, respectively, were given "G1," "G2," and "G3" genomic assignments. Median turnaround time was 7 days (IQR, 5-9). Response rates were 44.8%, 8.3%, 26.7%, and 55.6% for the "G1-SOX," "G1-SP," "G2," "G3" cohorts, respectively; and was higher in G1 patients treated with SOX compared with SP (P = 0.033). Exploratory analyses using the genomic classifier of Lei and colleagues validated the utility of the metabolic signature as a biomarker for predicting benefit from chemotherapy (log-rank P = 0.004 for PFS), whereas the Asian Cancer Research Group classifier did not demonstrate any predictive value.Conclusions: This bench-to-bedside effort establishes a reasonable turnaround time for gene expression profiling and possible utility of genomic classifiers in gastric cancer treatment stratification. Clin Cancer Res; 24(21); 5272-81. ©2018 AACR.

PD-L1 expression is mainly regulated by interferon gamma associated with JAK-STAT pathway in gastric cancer.

Despite multidisciplinary treatment for patients with advanced gastric cancer, their prognosis remains poor. Therefore, the development of novel therapeutic strategies is urgently needed, and immunotherapy utilizing anti-programmed death 1/-programmed death ligand-1 mAb is an attractive approach. However, as there is limited information on how programmed death ligand-1 is upregulated on tumor cells within the tumor microenvironment, we examined the mechanism of programmed death ligand-1 regulation with a particular focus on interferon gamma in an in vitro setting and in clinical samples. Our in vitro findings showed that interferon gamma upregulated programmed death ligand-1 expression on solid tumor cells through the JAK-signal transducer and activator of transcription pathway, and impaired the cytotoxicity of tumor antigen-specific CTL against tumor cells. Following treatment of cells with anti-programmed death ligand-1 mAb after interferon gamma-pre-treatment, the reduced anti-tumor CTL activity by interferon gamma reached a higher level than the non-treatment control targets. In contrast, programmed death ligand-1 expression on tumor cells also significantly correlated with epithelial-mesenchymal transition phenotype in a panel of solid tumor cells. In clinical gastric cancer samples, tumor membrane programmed death ligand-1 expression significantly positively correlated with the presence of CD8-positive T cells in the stroma and interferon gamma expression in the tumor. The results suggest that gastric cancer patients with high CD8-positive T-cell infiltration may be more responsive to anti-programmed death 1/-programmed death ligand-1 mAb therapy.

Accumulation of CD11c+CD163+ Adipose Tissue Macrophages through Upregulation of Intracellular 11ß-HSD1 in Human Obesity.

Adipose tissue (AT) macrophages (ATMs) are key players for regulation of AT homeostasis and obesity-related metabolic disorders. However, the phenotypes of human ATMs and regulatory mechanisms of their polarization have not been clearly described. In this study, we investigated human ATMs in both abdominal visceral AT and s.c. AT and proposed an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1)-glucocorticoid receptor regulatory axis that might dictate M1/M2 polarization in ATMs. The accumulation of CD11c+CD163+ ATMs in both visceral AT and s.c. AT of obese individuals was confirmed at the cellular level and was found to be clearly correlated with body mass index and production of reactive oxygen species. Using our in vitro system where human peripheral blood monocytes (hPBMs) were cocultured with Simpson-Golabi-Behmel syndrome adipocytes, M1/M2 polarization was found to be dependent on 11ß-HSD1, an intracellular glucocorticoid reactivating enzyme. Exposure of hPBMs to cortisol-induced expression of CD163 and RU-486, a glucocorticoid receptor antagonist, significantly abrogated CD163 expression through coculture of mature adipocytes with hPBMs. Moreover, 11ß-HSD1 was expressed in crown ATMs in obese AT. Importantly, conditioned medium from coculture of adipocytes with hPBMs enhanced proliferation of human breast cancer MCF7 and MDA-MB-231 cells. In summary, the phenotypic switch of ATMs from M2 to mixed M1/M2 phenotype occurred through differentiation of adipocytes in obese individuals, and upregulation of intracellular 11ß-HSD1 might play a role in the process.

Inhibition of MMP activity can restore NKG2D ligand expression in gastric cancer, leading to improved NK cell susceptibility.

BACKGROUND AND METHODS: Natural killer (NK) cells can react with tumor cells through the balance of inhibitory and stimulatory signals between NK cell surface receptors and their ligands, such as MHC class I chain-related A (MICA), MHC class I chain-related B (MICB), and several UL16-binding proteins (ULBPs). In the present study, we evaluated the relationship between NKG2D ligand expression and matrix metalloproteinase (MMP) activity in in vitro culture systems of a panel of gastric cancer cell lines (n = 10) and clinical samples (n = 102). RESULTS: First, the surface expression of NK group 2 member D (NKG2D) ligands (MICA, MICB, ULBP-2, and ULBP-3) on tumor cells was markedly downregulated on in vitro culture, in parallel to the upregulation of MMPs analyzed by gelatin zymography and gene expression microarray, whereas the transcript levels of NKG2D ligands remained unchanged on in vitro culture. Second, MMP-specific inhibitors could restore the downregulated expression of NKG2D ligands and functionally improve susceptibilities to NK cells in vitro. Third, the production of soluble NKG2D ligands was increased on in vitro culture and was inhibited by MMP-specific inhibitors. Finally, there was a significant inverse correlation between MMP-9 expression and NKG2D ligand expression as analyzed by immunohistochemistry in clinical tumor samples. CONCLUSION: The present study is a comprehensive study demonstrating that upregulation of MMP activity can induce a downregulation of expression of NKG2D ligands in gastric cancer cells, leading to lower-level susceptibility to NK cells.

Melanoma associated antigen (MAGE)-A3 promotes cell proliferation and chemotherapeutic drug resistance in gastric cancer.

BACKGROUND: Melanoma-associated antigen (MAGE)-A3 is a member of the family of cancer-testis antigens and has been found to be epigenetically regulated and aberrantly expressed in various cancer types. It has also been found that MAGE-A3 expression may correlate with an aggressive clinical course and with chemo-resistance. The objectives of this study were to assess the relationship between MAGE-A3 promoter methylation and expression and (1) gastric cancer patient survival and (2) its functional consequences in gastric cancer-derived cells. METHODS: Samples from two independent gastric cancer cohorts (including matched non-malignant gastric samples) were included in this study. MAGE-A3 methylation and mRNA expression levels were determined by methylation-specific PCR (MSP) and quantitative real-time PCR (qPCR), respectively. MAGE-A3 expression was knocked down in MKN1 gastric cancer-derived cells using miRNAs. In addition, in vitro cell proliferation, colony formation, apoptosis, cell cycle, drug treatment, immunohistochemistry and Western blot assays were performed. RESULTS: Clinical analysis of 223 primary patient-derived samples (ntumor = 161, nnormal = 62) showed a significant inverse correlation between MAGE-A3 promoter methylation and expression in the cancer samples (R = -0.63, p = 5.99e-19). A lower MAGE-A3 methylation level was found to be associated with a worse patient survival (HR: 1.5, 95 % CI: 1.02-2.37, p = 0.04). In addition, we found that miRNA-mediated knockdown of MAGE-A3 expression in MKN1 cells caused a reduction in its proliferation and colony forming capacities, respectively. Under stress conditions MAGE-A3 was found to regulate the expression of Bax and p21. MAGE-A3 knock down also led to an increase in Puma and Noxa expression, thus contributing to an enhanced docetaxel sensitivity in the gastric cancer-derived cells. CONCLUSIONS: From our results we conclude that MAGE-A3 expression is regulated epigenetically by promoter methylation, and that its expression contributes to gastric cell proliferation and drug sensitivity. This study underscores the potential implications of MAGE-A3 as a therapeutic target and prognostic marker in gastric cancer patients.

Development of a conditional liver tumor model by mifepristone-inducible Cre recombination to control oncogenic kras V12 expression in transgenic zebrafish.

Here we report a new transgenic expression system by combination of liver-specific expression, mifepristone induction and Cre-loxP recombination to conditionally control the expression of oncogenic kras(V12). This transgenic system allowed expression of kras(V12) specifically in the liver by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system. We found that liver tumors were generally induced from multiple foci due to incomplete Cre-loxP recombination, thus mimicking naturally occurring human tumors resulting from one or a few mutated cells and clonal proliferation to form nodules. Similar to our earlier studies by both constitutive and inducible expression of the kras(V12) oncogene, hepatocellular carcinoma (HCC) is the main type of liver tumor induced by kras(V12) expression. Moreover, mixed tumors with hepatocellular adenoma and hepatoblastoma (HB) were also frequently observed. Molecular analyses also indicated similar increase of phosphorylated ERK1/2 in all types of liver tumors, but nuclear localization of ß-catenin, a sign of malignant transformation, was found only in HCC and HB. Taken together, our new transgenic system reported in this study allows transgenic kras(V12) expression specifically in the zebrafish liver only by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system.

Knockdown of POLA2 increases gemcitabine resistance in lung cancer cells.

BACKGROUND: Gemcitabine is used as a standard drug treatment for non-small cell lung cancer (NSCLC), but treatment responses vary among patients. Our previous studies demonstrated that POLA2 + 1747 GG/GA single nucleotide polymorphism (SNP) improves differential survivability and mortality in NSCLC patients. Here, we determined the association between POLA2 and gemcitabine treatment in human lung cancer cells. RESULTS: Human PC9, H1299 and H1650 lung cancer cell lines were treated with 0.01-100 µM gemcitabine for 72 h. Although all 3 cell lines showed decreased cell viability upon gemcitabine treatment, H1299 was found to be the most sensitive to gemcitabine treatment. Next, sequencing was performed to determine if POLA2 + 1747 SNP might be involved in gemcitabine sensitivity. Data revealed that all 3 cell lines harbored the wild-type POLA2 + 1747 GG SNP, indicating that the POLA2 + 1747 SNP might not be responsible for gemcitabine sensitivity in the cell lines studied. Silencing of POLA2 gene in H1299 was then carried out by siRNA transfection, followed by gemcitabine treatment to determine the effect of POLA2 knockdown on chemosensitivity to gemcitabine. Results showed that H1299 exhibited increased resistance to gemcitabine after POLA2 knockdown, suggesting that POLA2 does not act alone and may cooperate with other interacting partners to cause gemcitabine resistance. CONCLUSIONS: Collectively, our findings showed that knockdown of POLA2 increases gemcitabine resistance in human lung cancer cells. We propose that POLA2 may play a role in gemcitabine sensitivity and can be used as a prognostic biomarker of patient outcome in NSCLC pathogenesis.