An investigation of the clinical impact and therapeutic relevance of a DNA damage immune response (DDIR) signature in patients with advanced gastroesophageal adenocarcinoma.

BACKGROUND: An improved understanding of which gastroesophageal adenocarcinoma (GOA) patients respond to both chemotherapy and immune checkpoint inhibitors (ICI) is needed. We investigated the predictive role and underlying biology of a 44-gene DNA damage immune response (DDIR) signature in patients with advanced GOA. MATERIALS AND METHODS: Transcriptional profiling was carried out on pretreatment tissue from 252 GOA patients treated with platinum-based chemotherapy (three dose levels) within the randomized phase III GO2 trial. Cross-validation was carried out in two independent GOA cohorts with transcriptional profiling, immune cell immunohistochemistry and epidermal growth factor receptor (EGFR) fluorescent in situ hybridization (FISH) (n = 430). RESULTS: In the GO2 trial, DDIR-positive tumours had a greater radiological response (51.7% versus 28.5%, P = 0.022) and improved overall survival in a dose-dependent manner (P = 0.028). DDIR positivity was associated with a pretreatment inflamed tumour microenvironment (TME) and increased expression of biomarkers associated with ICI response such as CD274 (programmed death-ligand 1, PD-L1) and a microsatellite instability RNA signature. Consensus pathway analysis identified EGFR as a potential key determinant of the DDIR signature. EGFR amplification was associated with DDIR negativity and an immune cold TME. CONCLUSIONS: Our results indicate the importance of the GOA TME in chemotherapy response, its relationship to DNA damage repair and EGFR as a targetable driver of an immune cold TME. Chemotherapy-sensitive inflamed GOAs could benefit from ICI delivered in combination with standard chemotherapy. Combining EGFR inhibitors and ICIs warrants further investigation in patients with EGFR-amplified tumours.

Melanoma secretion of transforming growth factor-ß2 leads to loss of epidermal AMBRA1 threatening epidermal integrity and facilitating tumour ulceration.

BACKGROUND: For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis. OBJECTIVES: The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-ß signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity. METHODS: Immunohistochemistry was used to analyse AMBRA1 and TGF-ß2 in a cohort of 109 AJCC all-stage melanomas, and TGF-ß2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-ß2 signalling evaluated in primary keratinocytes. RESULTS: Increased tumoral TGF-ß2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-ß2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05). CONCLUSIONS: Collectively, these data suggest a paracrine mechanism whereby TGF-ß2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration.

TGF-beta induces apoptosis in human B cells by transcriptional regulation of BIK and BCL-XL.

Transforming growth factor-beta (TGF-beta) potently induces apoptosis in Burkitt's lymphoma (BL) cell lines and in explanted primary human B lymphocytes. The physiological relevance and mechanism of TGF-beta-mediated apoptosis induction in these cells remains to be determined. Here we demonstrate the requirement for TGF-beta-mediated regulation of BIK and BCL-X(L) to activate an intrinsic apoptotic pathway in centroblastic BL cells. TGF-beta directly induced transcription of BIK and a consensus Smad-binding element identified in the BIK promoter recruits TGF-beta-activated Smad transcription factor complexes in vivo. TGF-beta also transcriptionally repressed expression of the apoptosis inhibitor BCL-X(L). Inhibition of BCL-X(L) sensitised BL cells to TGF-beta-induced apoptosis whereas overexpression of BCL-X(L) or suppression of BIK by shRNA, diminished TGF-beta-induced apoptosis. BIK and BCL-X(L) were also identified as TGF-beta target genes in purified normal human centroblast B cells and immunohistochemical analyses of tonsil tissue revealed widespread TGF-beta receptor-regulated Smad activation and a focal pattern of BIK expression. Furthermore, using a selective inhibitor of the TGF-beta receptor we provide evidence that autocrine TGF-beta signalling through ALK5 contributes to the default apoptotic programme in normal human centroblasts undergoing spontaneous apoptosis. Our data suggests that TGF-beta may act as a physiological mediator of human germinal centre homoeostasis by regulation of BIK and BCL-X(L).

TGF-beta-mediated activation of RhoA signalling is required for efficient (V12)HaRas and (V600E)BRAF transformation.

Transforming growth factor beta-1 (TGF-beta) acts as both a tumour suppressor and a tumour promoter in a context-dependent manner. The tumour-promoting activities of TGF-beta are likely to result from a combination of Smad and non-Smad signalling pathways but remain poorly understood. Here we show that TGF-beta-mediated activation of RhoA is dependent on the kinase activity of ALK5 and that continuous ALK5 activity maintains basal RhoA-ROCK signalling, cell morphology and actin dynamics in serum-starved rodent fibroblasts independently of Smad2, Smad3 and Smad4. In immortalized human diploid fibroblasts, we show that oncogenic rewiring by transduction of (V12)HaRas instigates regulation of RhoA-ROCK signalling through an autocrine TGF-beta1-ALK5 pathway. Furthermore, we show that ALK5-mediated activation of RhoA is required for efficient (V12)HaRas, V-Raf and (V600E)BRAF transformation and (V12)HaRas-mediated anchorage-independent growth. These findings identify a new pro-oncogenic activity of TGF-beta and indicate that tumours harbouring (V12)HaRas and (V600E)BRAF mutations may be susceptible to TGF-beta signalling inhibitors.

Direct and indirect regulation of cytokine and cell cycle proteins by EBNA-2 during Epstein-Barr virus infection.

We have studied the pathways of regulation of cytokine and cell cycle control proteins during infection of human B lymphocytes by Epstein-Barr virus (EBV). Among 30 cytokine RNAs analyzed by the RNase protection assay, tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor, lymphotoxin (LT), and LTbeta were found to be regulated within 20 h of EBV infection of primary B cells. Similar results were obtained using the estrogen-regulated EBNA-2 cell line EREB2.5, in which RNAs for LT and TNF-alpha were induced within 6 h of activation of EBNA-2. Expression of Notch also caused an induction of TNF-alpha RNA. The induction of TNF-alpha RNA by EBNA-2 was indirect, and constitutive expression of either LMP-1 or c-myc proteins did not substitute for EBNA-2 in induction of TNF-alpha RNA. Cyclin D2 is also an indirect target of EBNA-2-mediated transactivation. EBNA-2 was found to activate the cyclin D2 promoter in a transient-transfection assay. A mutant of EBNA-2 that does not bind RBP-Jkappa retained some activity in this assay, and activation did not depend on the presence of B-cell-specific factors. Deletion analysis of the cyclin D2 promoter revealed that removal of sequences containing E-box c-myc consensus DNA binding sequences did not reduce EBNA-2-mediated activation of the cyclin D2 promoter in the transient-transfection assay. The results indicate that cytokines are an early target of EBNA-2 and that EBNA-2 can regulate cyclin D2 transcription in EBV-infected cells by mechanisms additional to the c-myc pathway.

Control of cell cycle entry and apoptosis in B lymphocytes infected by Epstein-Barr virus.

Infection of human B cells with Epstein-Barr virus (EBV) results in activation of the cell cycle and cell growth. To interpret the mechanisms by which EBV activates the cell, we have assayed many proteins involved in control of the G0 and G1 phases of the cell cycle and regulation of apoptosis. In EBV infection most of the changes, including the early induction of cyclin D2, are dependent on expression of EBV genes, but an alteration in the E2F-4 profile was partly independent of viral gene expression, presumably occurring in response to signal transduction activated when the virus binds to its receptor, CD21. By comparing the expression of genes controlling apoptosis, including those encoding several members of the BCL-2 family of proteins, the known relative resistance of EBV-immortalized B-cell lines to apoptosis induced by low serum was found to correlate with expression of both BCL-2 and A20. A20 can be regulated by the NF-kappaB transcription factor, which is known to be activated by the EBV LMP-1 protein. Quantitative assays demonstrated a direct temporal relationship between LMP-1 protein levels and active NF-kappaB during the time course of infection.

Abundant IFN-gamma production by local T cells in respiratory syncytial virus-induced eosinophilic lung disease.

Respiratory syncytial virus (RSV) is a frequent cause of severe lung disease in young children. Primed T cells are required for virus clearance, but are causally implicated in the enhanced pathology seen following RSV infection of some infants and experimental animals vaccinated against the virus. In BALB/c mice, vaccination with recombinant vaccinia virus expressing the viral attachment protein (G) leads to pulmonary eosinophilia during subsequent infection, which indirect evidence suggests may be due to CD4+ Th2 cells. The production of IFN-gamma, IL-2, -4, -5 and -10 cytokine mRNA by RT-PCR and intracellular cytokines by flow cytometry following RSV challenge of vaccinated mice were therefore compared. Lung eosinophilia was associated with enhanced local recruitment of CD4+ cells in G sensitized mice, while CD8+ cells dominated in mice vaccinated with the viral fusion protein (F) or second matrix protein (M2). Lung eosinophilia was also associated with a localized reduction in IFN-gamma and increased IL-4 and IL-5 mRNA transcription as well as elevated RSV specific IgG1 antibody production. Th2 cytokine protein production by T cells showed no apparent change. Although IFN-gamma production diminished in eosinophilic mice, it remained the major cytokine found in lung T cells. It was concluded that lung eosinophilia can develop despite abundant IFN-gamma production by local T cells, but is associated with a shift in the balance between Th2 and Th1 cytokine production.

Th1 and Th2 cytokine induction in pulmonary T cells during infection with respiratory syncytial virus.

Helper T (Th) cells can be classified functionally into two main types. Broadly, Th1 cells play a major role in eliminating viral pathogens, while Th2 cells mediate anti-parasite immunity and allergic responses. These functions are thought to depend on characteristic and distinct patterns of cytokine production. Infection with human respiratory syncytial virus, an important common cold virus, causes transient lymphocytic bronchiolitis in mice. Activated T cells are partly responsible for this disease, but also eliminate the virus. To show whether polarized cytokine production occurs in individual cells during viral bronchiolitis, we sampled murine bronchoalveolar lavage and mediastinal lymph node cells before and after infection. RT-PCR of cellular mRNA and flow cytometric analysis of intracellular cytokine production showed a rapid IFN-gamma response at both sites, which persisted for more than 3 weeks in the lung. Most IFN-gamma-producing cells were CD8+. Some early CD4+ IFN-gamma-producing cells also made IL-10. Only low levels of IL-2, IL-4 and IL-5 mRNA or protein expression were detected at any time at either site. No cytokines were detected in B cell populations at either site. These novel techniques show the true complexity of cytokine production patterns on a cell-by-cell basis, allowing T cells to be reclassified according to function.

Activity of penciclovir in antiviral assays against herpes simplex virus.

The effect of penciclovir and acyclovir on the replication of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains was determined in MRC-5 cells infected with 0.01 pfu/cell and exposed to the drugs for 72 h to allow multiple cycles of replication. Penciclovir was significantly more active than acyclovir against three strains of HSV-1 and three strains of HSV-2 at 1 mg/L (P = 0.009), 3 mg/L (P < 0.001) and 10 mg/L (P = 0.001). Further comparisons between the compounds were made in MRC-5 cells infected with HSV-1 strain SC16 using four different antiviral assays namely, the 24 h virus yield reduction assay, plaque reduction assay, viral antigen inhibition assay, and a viral DNA inhibition assay, to determine the relative merits of each. Penciclovir and acyclovir shared similar activities in the plaque reduction assay (with 50% effective concentrations, EC50, being 0.8 and 0.6 mg/L, respectively) and in the viral antigen inhibition assay (EC50s. 0.6 and 0.7 mg/L, respectively). The EC50 of penciclovir in the 24 h viral DNA inhibition assay was 0.01 mg/L compared with 0.06 mg/L of acyclovir. In the 24 h virus yield reduction assay in which MRC-5 cells were infected with 0.3 pfu/cell, penciclovir was more active than acyclovir with 99% effective concentrations of 0.6 mg/L and 1.1 mg/L, respectively. The activity of penciclovir in the 24 h virus yield reduction and antigen inhibition assays was inversely related to the multiplicity of infection, whereas this had considerably less effect on the inhibition of viral DNA synthesis. These results suggest that famciclovir, which is the oral form of penciclovir, will be at least as effective as acyclovir in treating infections caused by HSV-1 and HSV-2.