Methionine restriction-induced sulfur deficiency impairs antitumour immunity partially through gut microbiota.

Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.

DNASE1L3 enhances antitumor immunity and suppresses tumor progression in colon cancer.

DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.

Predicting tumor response to drugs based on gene-expression biomarkers of sensitivity learned from cancer cell lines.

BACKGROUND: Human cancer cell line profiling and drug sensitivity studies provide valuable information about the therapeutic potential of drugs and their possible mechanisms of action. The goal of those studies is to translate the findings from in vitro studies of cancer cell lines into in vivo therapeutic relevance and, eventually, patients' care. Tremendous progress has been made. RESULTS: In this work, we built predictive models for 453 drugs using data on gene expression and drug sensitivity (IC50) from cancer cell lines. We identified many known drug-gene interactions and uncovered several potentially novel drug-gene associations. Importantly, we further applied these predictive models to ~ 17,000 bulk RNA-seq samples from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database to predict drug sensitivity for both normal and tumor tissues. We created a web site for users to visualize and download our predicted data ( https://manticore.niehs.nih.gov/cancerRxTissue ). Using trametinib as an example, we showed that our approach can faithfully recapitulate the known tumor specificity of the drug. CONCLUSIONS: We demonstrated that our approach can predict drugs that 1) are tumor-type specific; 2) elicit higher sensitivity from tumor compared to corresponding normal tissue; 3) elicit differential sensitivity across breast cancer subtypes. If validated, our prediction could have relevance for preclinical drug testing and in phase I clinical design.

HNF4α regulates sulfur amino acid metabolism and confers sensitivity to methionine restriction in liver cancer.

Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. Here we report that hepatocyte nuclear factor 4α (HNF4α) dictates the sensitivity of liver cancer to methionine restriction. We show that hepatic sulfur amino acid (SAA) metabolism is under transcriptional control of HNF4α. Knocking down HNF4α or SAA enzymes in HNF4α-positive epithelial liver cancer lines impairs SAA metabolism, increases resistance to methionine restriction or sorafenib, promotes epithelial-mesenchymal transition, and induces cell migration. Conversely, genetic or metabolic restoration of the transsulfuration pathway in SAA metabolism significantly alleviates the outcomes induced by HNF4α deficiency in liver cancer cells. Our study identifies HNF4α as a regulator of hepatic SAA metabolism that regulates the sensitivity of liver cancer to methionine restriction.

Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

CDSeq: A novel complete deconvolution method for dissecting heterogeneous samples using gene expression data.

Quantifying cell-type proportions and their corresponding gene expression profiles in tissue samples would enhance understanding of the contributions of individual cell types to the physiological states of the tissue. Current approaches that address tissue heterogeneity have drawbacks. Experimental techniques, such as fluorescence-activated cell sorting, and single cell RNA sequencing are expensive. Computational approaches that use expression data from heterogeneous samples are promising, but most of the current methods estimate either cell-type proportions or cell-type-specific expression profiles by requiring the other as input. Although such partial deconvolution methods have been successfully applied to tumor samples, the additional input required may be unavailable. We introduce a novel complete deconvolution method, CDSeq, that uses only RNA-Seq data from bulk tissue samples to simultaneously estimate both cell-type proportions and cell-type-specific expression profiles. Using several synthetic and real experimental datasets with known cell-type composition and cell-type-specific expression profiles, we compared CDSeq's complete deconvolution performance with seven other established deconvolution methods. Complete deconvolution using CDSeq represents a substantial technical advance over partial deconvolution approaches and will be useful for studying cell mixtures in tissue samples. CDSeq is available at GitHub repository (MATLAB and Octave code): https://github.com/kkang7/CDSeq.

Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma.

Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.

Expression level is a key determinant of E2F1-mediated cell fate.

The Rb/E2F network has a critical role in regulating cell cycle progression and cell fate decisions. It is dysfunctional in virtually all human cancers, because of genetic lesions that cause overexpression of activators, inactivation of repressors, or both. Paradoxically, the downstream target of this network, E2F1, is rarely strongly overexpressed in cancer. E2F1 can induce both proliferation and apoptosis but the factors governing these critical cell fate decisions remain unclear. Previous studies have focused on qualitative mechanisms such as differential cofactors, posttranslational modification or state of other signaling pathways as modifiers of the cell fate decisions downstream of E2F1 activation. In contrast, the importance of the expression levels of E2F1 itself in dictating the downstream phenotypes has not been rigorously studied, partly due to the limited resolution of traditional population-level measurements. Here, through single-cell quantitative analysis, we demonstrate that E2F1 expression levels have a critical role in determining the fate of individual cells. Low levels of exogenous E2F1 promote proliferation, moderate levels induce G1, G2 and mitotic cell cycle arrest, and very high levels promote apoptosis. These multiple anti-proliferative mechanisms result in a strong selection pressure leading to rapid elimination of E2F1-overexpressing cells from the population. RNA-sequencing and RT-PCR revealed that low levels of E2F1 are sufficient to induce numerous cell cycle-promoting genes, intermediate levels induce growth arrest genes (i.e., p18, p19 and p27), whereas higher levels are necessary to induce key apoptotic E2F1 targets APAF1, PUMA, HRK and BIM. Finally, treatment of a lung cancer cell line with a proteasome inhibitor, MLN2238, resulted in an E2F1-dependent mitotic arrest and apoptosis, confirming the role of endogenous E2F1 levels in these phenotypes. The strong anti-proliferative activity of moderately overexpressed E2F1 in multiple cancer types suggests that targeting E2F1 for upregulation may represent an attractive therapeutic strategy in cancer.

p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways.

The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NFκB subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNFα, another activator of NFκB and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment.

E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

FOXO transcription factors control E2F1 transcriptional specificity and apoptotic function.

The transcription factor E2F1 is a key regulator of proliferation and apoptosis but the molecular mechanisms that mediate these cell fate decisions remain unclear. Here, we identify FOXO transcription factors as E2F1 target genes that act in a feed-forward regulatory loop to reinforce gene induction of multiple apoptotic genes. We found that E2F1 forms a complex with FOXO1 and FOXO3. RNAi-mediated silencing of FOXO impaired E2F1 binding to the promoters of cooperative target genes. A FOXO3 mutant insensitive to inactivation by survival kinases rescued the inhibitory effect of growth factor signaling on E2F1-mediated transcription and apoptosis. The E2F1/FOXO axis is frequently blocked in cancer, as evidenced by the specific downregulation of the FOXO-dependent E2F1 transcriptional program in multiple cancer types and by the association of a reduced E2F1/FOXO transcriptional program with poor prognosis. HDAC and phosphoinositide 3-kinase (PI3K) inhibitors were identified as specific activators of E2F1/FOXO transcription, acting to enhance E2F1-induced apoptosis in a FOXO3-dependent manner. Notably, combining the histone deacetylase inhibitor vorinostat with a PI3K inhibitor led to enhanced FOXO-dependent apoptosis. Collectively, our results identify E2F1/FOXO cooperation as a regulatory mechanism that places E2F1 apoptotic activity under the control of survival signaling. Therapeutic reactivation of this tumor suppressive mechanism may offer a novel broad-acting therapy for cancer.

Interaction of E2F7 transcription factor with E2F1 and C-terminal-binding protein (CtBP) provides a mechanism for E2F7-dependent transcription repression.

Previous work has identified distinct functions for E2F proteins during a cellular proliferative response including a role for E2F1-3 in the activation of transcription at G1/S and a role for E2F4-8 in repressing the same group of E2F1-3 target genes as cells progress through S phase. We now find that E2F7 and E2F8, which are induced by E2F1-3 at G1/S, can form a heterodimer with E2F1 through interactions involving the DNA-binding domains of the two proteins. In vitro DNA interaction assays demonstrate the formation of an E2F1-E2F7 complex, as well as an E2F7-E2F7 complex on adjacent E2F-binding sites. We also show that E2F7 recruits the co-repressor C-terminal-binding protein (CtBP) and that CtBP2 is essential for E2F7 to repress E2F1 transcription. Taken together, these findings suggest a mechanism for the repression of transcription by E2F7.

Using a stem cell-based signature to guide therapeutic selection in cancer.

Given the very substantial heterogeneity of most human cancers, it is likely that most cancer therapeutics will be active in only a small fraction of any population of patients. As such, the development of new therapeutics, coupled with methods to match a therapy with the individual patient, will be critical to achieving significant gains in disease outcome. One such opportunity is the use of expression signatures to identify key oncogenic phenotypes that can serve not only as biomarkers but also as a means of identifying therapeutic compounds that might specifically target these phenotypes. Given the potential importance of targeting tumors exhibiting a stem-like phenotype, we have developed an expression signature that reflects common biological aspects of various stem-like characteristics. The consensus stemness ranking (CSR) signature is upregulated in cancer stem cell-enriched samples at advanced tumor stages and is associated with poor prognosis in multiple cancer types. Using two independent computational approaches we utilized the CSR signature to identify clinically useful compounds that could target the CSR phenotype. In vitro assays confirmed selectivity of several predicted compounds including topoisomerase inhibitors and resveratrol towards breast cancer cell lines that exhibit a high-CSR phenotype. Importantly, the CSR signature could predict clinical response of breast cancer patients to a neoadjuvant regimen that included a CSR-specific agent. Collectively, these results suggest therapeutic opportunities to target the CSR phenotype in a relevant cohort of cancer patients.

p53 plays a role in mesenchymal differentiation programs, in a cell fate dependent manner.

BACKGROUND: The tumor suppressor p53 is an important regulator that controls various cellular networks, including cell differentiation. Interestingly, some studies suggest that p53 facilitates cell differentiation, whereas others claim that it suppresses differentiation. Therefore, it is critical to evaluate whether this inconsistency represents an authentic differential p53 activity manifested in the various differentiation programs. METHODOLOGY/PRINCIPAL FINDINGS: To clarify this important issue, we conducted a comparative study of several mesenchymal differentiation programs. The effects of p53 knockdown or enhanced activity were analyzed in mouse and human mesenchymal cells, representing various stages of several differentiation programs. We found that p53 down-regulated the expression of master differentiation-inducing transcription factors, thereby inhibiting osteogenic, adipogenic and smooth muscle differentiation of multiple mesenchymal cell types. In contrast, p53 is essential for skeletal muscle differentiation and osteogenic re-programming of skeletal muscle committed cells. CONCLUSIONS: These comparative studies suggest that, depending on the specific cell type and the specific differentiation program, p53 may exert a positive or a negative effect, and thus can be referred as a "guardian of differentiation" at large.

Myocardin in tumor suppression and myofibroblast differentiation.

The malignant transformation process is associated with defects in cell cycle regulation and disruption of the normal differentiation programs in both neoplastic and adjacent stroma cells. However, the relationships between the cell cycle, differentiation and cancer are very complex and tissue specific. Recently we have demonstrated a previously unrecognized role in human carcinogenesis for the important regulator of cardiac and smooth muscle differentiation, myocardin. Myocardin expression is frequently repressed during human malignant transformation contributing to a differentiation defect in the premalignant mesenchymal cells. TGFbeta treatment, serum deprivation and intact contact inhibition response all contribute to myocardin induction and differentiation. Positive regulation of myocardin mRNA levels and activity by the p16/Rb pathway provides a molecular link between cell cycle and differentiation defects during cancer development. In addition, we show that myocardin represses its own expression in human fibroblasts. This negative autoregulatory loop might be potentially important for restraining myocardin activity and allowing reversibility of fibroblast-myofibroblast phenotypic conversion. Here we discuss the emerging role of myocardin in tumor suppression as well as novel aspects of its regulation in normal and malignant conditions.

Inactivation of myocardin and p16 during malignant transformation contributes to a differentiation defect.

Myocardin is known as an important transcriptional regulator in smooth and cardiac muscle development. Here we found that myocardin is frequently repressed during human malignant transformation, contributing to a differentiation defect. We demonstrate that myocardin is a transcriptional target of TGFbeta required for TGFbeta-mediated differentiation of human fibroblasts. Serum deprivation, intact contact inhibition response, and the p16ink4a/Rb pathway contribute to myocardin induction and differentiation. Restoration of myocardin expression in sarcoma cells results in differentiation and inhibition of malignant growth, whereas inactivation of myocardin in normal fibroblasts increases their proliferative potential. Myocardin expression is reduced in multiple types of human tumors. Collectively, our results demonstrate that myocardin is an important suppressive modifier of the malignant transformation process.

Mutant p53 protects cells from 12-O-tetradecanoylphorbol-13-acetate-induced death by attenuating activating transcription factor 3 induction.

Mutations in p53 are ubiquitous in human tumors. Some p53 mutations not only result in loss of wild-type (WT) activity but also grant additional functions, termed "gain of function." In this study, we explore how the status of p53 affects the immediate response gene activating transcription factor 3 (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-protein kinase C (PKC) pathway. We show that high doses of TPA induce ATF3 in a WT p53-independent manner correlating with PKCs depletion and cell death. We show that cells harboring mutant p53 have attenuated ATF3 induction and are less sensitive to TPA-induced death compared with their p53-null counterparts. Mutagenesis analysis of the ATF3 promoter identified the regulatory motifs cyclic AMP-responsive element binding protein/ATF and MEF2 as being responsible for the TPA-induced activation of ATF3. Moreover, we show that mutant p53 attenuates ATF3 expression by two complementary mechanisms. It interacts with the ATF3 promoter and influences its activity via the MEF2 site, and additionally, it attenuates transcriptional expression of the ATF3 activator MEF2D. These data provide important insights into the molecular mechanisms that underlie mutant p53 gain of function.

hTERT-immortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis.

Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.

Transcriptional programs following genetic alterations in p53, INK4A, and H-Ras genes along defined stages of malignant transformation.

The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.