Disruption of metazoan gene regulatory networks in cancer alters the balance of co-expression between genes of unicellular and multicellular origins.

BACKGROUND: Metazoans inherited genes from unicellular ancestors that perform essential biological processes such as cell division, metabolism, and protein translation. Multicellularity requires careful control and coordination of these unicellular genes to maintain tissue integrity and homeostasis. Gene regulatory networks (GRNs) that arose during metazoan evolution are frequently altered in cancer, resulting in over-expression of unicellular genes. We propose that an imbalance in co-expression of unicellular (UC) and multicellular (MC) genes is a driving force in cancer. RESULTS: We combine gene co-expression analysis to infer changes to GRNs in cancer with protein sequence conservation data to distinguish genes with UC and MC origins. Co-expression networks created using RNA sequencing data from 31 tumor types and normal tissue samples are divided into modules enriched for UC genes, MC genes, or mixed UC-MC modules. The greatest differences between tumor and normal tissue co-expression networks occur within mixed UC-MC modules. MC and UC genes not commonly co-expressed in normal tissues form distinct co-expression modules seen only in tumors. The degree of rewiring of genes within mixed UC-MC modules increases with tumor grade and stage. Mixed UC-MC modules are enriched for somatic mutations in cancer genes, particularly amplifications, suggesting an important driver of the rewiring observed in tumors is copy number changes. CONCLUSIONS: Our study shows the greatest changes to gene co-expression patterns during tumor progression occur between genes of MC and UC origins, implicating the breakdown and rewiring of metazoan gene regulatory networks in cancer development and progression.

Spatial analysis with SPIAT and spaSim to characterize and simulate tissue microenvironments.

Spatial proteomics technologies have revealed an underappreciated link between the location of cells in tissue microenvironments and the underlying biology and clinical features, but there is significant lag in the development of downstream analysis methods and benchmarking tools. Here we present SPIAT (spatial image analysis of tissues), a spatial-platform agnostic toolkit with a suite of spatial analysis algorithms, and spaSim (spatial simulator), a simulator of tissue spatial data. SPIAT includes multiple colocalization, neighborhood and spatial heterogeneity metrics to characterize the spatial patterns of cells. Ten spatial metrics of SPIAT are benchmarked using simulated data generated with spaSim. We show how SPIAT can uncover cancer immune subtypes correlated with prognosis in cancer and characterize cell dysfunction in diabetes. Our results suggest SPIAT and spaSim as useful tools for quantifying spatial patterns, identifying and validating correlates of clinical outcomes and supporting method development.

Cystathionine-ß-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-ß-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

Reprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our previous first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single-agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.

A functional genetic screen defines the AKT-induced senescence signaling network.

Exquisite regulation of PI3K/AKT/mTORC1 signaling is essential for homeostatic control of cell growth, proliferation, and survival. Aberrant activation of this signaling network is an early driver of many sporadic human cancers. Paradoxically, sustained hyperactivation of the PI3K/AKT/mTORC1 pathway in nontransformed cells results in cellular senescence, which is a tumor-suppressive mechanism that must be overcome to promote malignant transformation. While oncogene-induced senescence (OIS) driven by excessive RAS/ERK signaling has been well studied, little is known about the mechanisms underpinning the AKT-induced senescence (AIS) response. Here, we utilize a combination of transcriptome and metabolic profiling to identify key signatures required to maintain AIS. We also employ a whole protein-coding genome RNAi screen for AIS escape, validating a subset of novel mediators and demonstrating their preferential specificity for AIS as compared with OIS. As proof of concept of the potential to exploit the AIS network, we show that neurofibromin 1 (NF1) is upregulated during AIS and its ability to suppress RAS/ERK signaling facilitates AIS maintenance. Furthermore, depletion of NF1 enhances transformation of p53-mutant epithelial cells expressing activated AKT, while its overexpression blocks transformation by inducing a senescent-like phenotype. Together, our findings reveal novel mechanistic insights into the control of AIS and identify putative senescence regulators that can potentially be targeted, with implications for new therapeutic options to treat PI3K/AKT/mTORC1-driven cancers.

Somatic mutations in early metazoan genes disrupt regulatory links between unicellular and multicellular genes in cancer.

Extensive transcriptional alterations are observed in cancer, many of which activate core biological processes established in unicellular organisms or suppress differentiation pathways formed in metazoans. Through rigorous, integrative analysis of genomics data from a range of solid tumors, we show many transcriptional changes in tumors are tied to mutations disrupting regulatory interactions between unicellular and multicellular genes within human gene regulatory networks (GRNs). Recurrent point mutations were enriched in regulator genes linking unicellular and multicellular subnetworks, while copy-number alterations affected downstream target genes in distinctly unicellular and multicellular regions of the GRN. Our results depict drivers of tumourigenesis as genes that created key regulatory links during the evolution of early multicellular life, whose dysfunction creates widespread dysregulation of primitive elements of the GRN. Several genes we identified as important in this process were associated with drug response, demonstrating the potential clinical value of our approach.

How the evolution of multicellularity set the stage for cancer.

Neoplastic growth and many of the hallmark properties of cancer are driven by the disruption of molecular networks established during the emergence of multicellularity. Regulatory pathways and molecules that evolved to impose regulatory constraints upon networks established in earlier unicellular organisms enabled greater communication and coordination between the diverse cell types required for multicellularity, but also created liabilities in the form of points of vulnerability in the network that when mutated or dysregulated facilitate the development of cancer. These factors are usually overlooked in genomic analyses of cancer, but understanding where vulnerabilities to cancer lie in the networks of multicellular species would provide important new insights into how core molecular processes and gene regulation change during tumourigenesis. We describe how the evolutionary origins of genes influence their roles in cancer, and how connections formed between unicellular and multicellular genes that act as key regulatory hubs for normal tissue homeostasis can also contribute to malignant transformation when disrupted. Tumours in general are characterised by increased dependence on unicellular processes for survival, and major dysregulation of the control structures imposed on these processes during the evolution of multicellularity. Mounting molecular evidence suggests altered interactions at the interface between unicellular and multicellular genes play key roles in the initiation and progression of cancer. Furthermore, unicellular network regions activated in cancer show high degrees of robustness and plasticity, conferring increased adaptability to tumour cells by supporting effective responses to environmental pressures such as drug exposure. Examining how the links between multicellular and unicellular regions get disrupted in tumours has great potential to identify novel drivers of cancer, and to guide improvements to cancer treatment by identifying more effective therapeutic strategies. Recent successes in targeting unicellular processes by novel compounds underscore the logic of such approaches. Further gains could come from identifying genes at the interface between unicellular and multicellular processes and manipulating the communication between network regions of different evolutionary ages.

Altered interactions between unicellular and multicellular genes drive hallmarks of transformation in a diverse range of solid tumors.

Tumors of distinct tissues of origin and genetic makeup display common hallmark cellular phenotypes, including sustained proliferation, suppression of cell death, and altered metabolism. These phenotypic commonalities have been proposed to stem from disruption of conserved regulatory mechanisms evolved during the transition to multicellularity to control fundamental cellular processes such as growth and replication. Dating the evolutionary emergence of human genes through phylostratigraphy uncovered close association between gene age and expression level in RNA sequencing data from The Cancer Genome Atlas for seven solid cancers. Genes conserved with unicellular organisms were strongly up-regulated, whereas genes of metazoan origin were primarily inactivated. These patterns were most consistent for processes known to be important in cancer, implicating both selection and active regulation during malignant transformation. The coordinated expression of strongly interacting multicellularity and unicellularity processes was lost in tumors. This separation of unicellular and multicellular functions appeared to be mediated by 12 highly connected genes, marking them as important general drivers of tumorigenesis. Our findings suggest common principles closely tied to the evolutionary history of genes underlie convergent changes at the cellular process level across a range of solid cancers. We propose altered activity of genes at the interfaces between multicellular and unicellular regions of human gene regulatory networks activate primitive transcriptional programs, driving common hallmark features of cancer. Manipulation of cross-talk between biological processes of different evolutionary origins may thus present powerful and broadly applicable treatment strategies for cancer.