Cancer Evolution: A Multifaceted Affair.

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery. SIGNIFICANCE: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution.

The SWI/SNF complex member SMARCB1 supports lineage fidelity in kidney cancer.

Lineage switching can induce therapy resistance in cancer. Yet, how lineage fidelity is maintained and how it can be lost remain poorly understood. Here, we have used CRISPR-Cas9-based genetic screening to demonstrate that loss of SMARCB1, a member of the SWI/SNF chromatin remodeling complex, can confer an advantage to clear cell renal cell carcinoma (ccRCC) cells upon inhibition of the renal lineage factor PAX8. Lineage factor inhibition-resistant ccRCC cells formed tumors with morphological features, but not molecular markers, of neuroendocrine differentiation. SMARCB1 inactivation led to large-scale loss of kidney-specific epigenetic programs and restoration of proliferative capacity through the adoption of new dependencies on factors that represent rare essential genes across different cancers. We further developed an analytical approach to systematically characterize lineage fidelity using large-scale CRISPR-Cas9 data. An understanding of the rules that govern lineage switching could aid the development of more durable lineage factor-targeted and other cancer therapies.

Cancer Cell-Derived PDGFB Stimulates mTORC1 Activation in Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a hypervascular tumor that is characterized by bi-allelic inactivation of the VHL tumor suppressor gene and mTOR signalling pathway hyperactivation. The pro-angiogenic factor PDGFB, a transcriptional target of super enhancer-driven KLF6, can activate the mTORC1 signalling pathway in ccRCC. However, the detailed mechanisms of PDGFB-mediated mTORC1 activation in ccRCC have remained elusive. Here, we investigated whether ccRCC cells are able to secrete PDGFB into the extracellular milieu and stimulate mTORC1 signalling activity. We found that ccRCC cells secreted PDGFB extracellularly, and by utilizing KLF6- and PDGFB-engineered ccRCC cells, we showed that the level of PDGFB secretion was positively correlated with the expression of intracellular KLF6 and PDGFB. Moreover, the reintroduction of either KLF6 or PDGFB was able to sustain mTORC1 signalling activity in KLF6-targeted ccRCC cells. We further demonstrated that conditioned media of PDGFB-overexpressing ccRCC cells was able to re-activate mTORC1 activity in KLF6-targeted cells. In conclusion, cancer cell-derived PDGFB can mediate mTORC1 signalling pathway activation in ccRCC, further consolidating the link between the KLF6-PDGFB axis and the mTORC1 signalling pathway activity in ccRCC.

Dynamic partitioning of branched-chain amino acids-derived nitrogen supports renal cancer progression.

Metabolic reprogramming is critical for tumor initiation and progression. However, the exact impact of specific metabolic changes on cancer progression is poorly understood. Here, we integrate multimodal analyses of primary and metastatic clonally-related clear cell renal cancer cells (ccRCC) grown in physiological media to identify key stage-specific metabolic vulnerabilities. We show that a VHL loss-dependent reprogramming of branched-chain amino acid catabolism sustains the de novo biosynthesis of aspartate and arginine enabling tumor cells with the flexibility of partitioning the nitrogen of the amino acids depending on their needs. Importantly, we identify the epigenetic reactivation of argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary ccRCC, as a crucial event for metastatic renal cancer cells to acquire the capability to generate arginine, invade in vitro and metastasize in vivo. Overall, our study uncovers a mechanism of metabolic flexibility occurring during ccRCC progression, paving the way for the development of novel stage-specific therapies.

HIRA loss transforms FH-deficient cells.

Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.].

Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma.

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

Genomic control of metastasis.

Metastasis remains the leading cause of cancer-associated mortality, and a detailed understanding of the metastatic process could suggest new therapeutic avenues. However, how metastatic phenotypes arise at the genomic level has remained a major open question in cancer biology. Comparative genetic studies of primary and metastatic cancers have revealed a complex picture of metastatic evolution with diverse temporal patterns and trajectories to dissemination. Whole-genome amplification is associated with metastatic cancer clones, but no metastasis-exclusive driver mutations have emerged. Instead, genetically activated oncogenic pathways that drive tumour initiation and early progression acquire metastatic traits by co-opting physiological programmes from stem cell, developmental and regenerative pathways. The functional consequences of oncogenic driver mutations therefore change via epigenetic mechanisms to promote metastasis. Increasing evidence is starting to uncover the molecular mechanisms that determine how specific oncogenic drivers interact with various physiological programmes, and what triggers their activation in support of metastasis. Detailed insight into the mechanisms that control metastasis is likely to reveal novel opportunities for intervention at different stages of metastatic progression.

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism.

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.

A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma.

Transcriptional networks are critical for the establishment of tissue-specific cellular states in health and disease, including cancer. Yet, the transcriptional circuits that control carcinogenesis remain poorly understood. Here we report that Kruppel like factor 6 (KLF6), a transcription factor of the zinc finger family, regulates lipid homeostasis in clear cell renal cell carcinoma (ccRCC). We show that KLF6 supports the expression of lipid metabolism genes and promotes the expression of PDGFB, which activates mTOR signalling and the downstream lipid metabolism regulators SREBF1 and SREBF2. KLF6 expression is driven by a robust super enhancer that integrates signals from multiple pathways, including the ccRCC-initiating VHL-HIF2A pathway. These results suggest an underlying mechanism for high mTOR activity in ccRCC cells. More generally, the link between super enhancer-driven transcriptional networks and essential metabolic pathways may provide clues to the mechanisms that maintain the stability of cell identity-defining transcriptional programmes in cancer.

Circulating Tumor Cells: Come Together, Right Now, Over Metastasis.

Circulating tumor cells (CTC) are the source of metastases, but only an infinitesimal fraction of them eventually succeed in colonizing a distant organ. New results show that CD44-dependent aggregation in the circulation provides CTCs with cancer stem cell-like characteristics, suggesting an explanation for the low metastatic efficiency of CTCs, but also avenues for therapeutic intervention.See related article by Liu et al., p. 96.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

Endogenous HIF2A reporter systems for high-throughput functional screening.

Tissue-specific transcriptional programs control most biological phenotypes, including disease states such as cancer. However, the molecular details underlying transcriptional specificity is largely unknown, hindering the development of therapeutic approaches. Here, we describe novel experimental reporter systems that allow interrogation of the endogenous expression of HIF2A, a critical driver of renal oncogenesis. Using a focused CRISPR-Cas9 library targeting chromatin regulators, we provide evidence that these reporter systems are compatible with high-throughput screening. Our data also suggests redundancy in the control of cancer type-specific transcriptional traits. Reporter systems such as those described here could facilitate large-scale mechanistic dissection of transcriptional programmes underlying cancer phenotypes, thus paving the way for novel therapeutic approaches.

NF-κB-Dependent Lymphoid Enhancer Co-option Promotes Renal Carcinoma Metastasis.

Metastases, the spread of cancer cells to distant organs, cause the majority of cancer-related deaths. Few metastasis-specific driver mutations have been identified, suggesting aberrant gene regulation as a source of metastatic traits. However, how metastatic gene expression programs arise is poorly understood. Here, using human-derived metastasis models of renal cancer, we identify transcriptional enhancers that promote metastatic carcinoma progression. Specific enhancers and enhancer clusters are activated in metastatic cancer cell populations, and the associated gene expression patterns are predictive of poor patient outcome in clinical samples. We find that the renal cancer metastasis-associated enhancer complement consists of multiple coactivated tissue-specific enhancer modules. Specifically, we identify and functionally characterize a coregulatory enhancer cluster, activated by the renal cancer driver HIF2A and an NF-κB-driven lymphoid element, as a mediator of metastasis in vivo We conclude that oncogenic pathways can acquire metastatic phenotypes through cross-lineage co-option of physiologic epigenetic enhancer states.Significance: Renal cancer is associated with significant mortality due to metastasis. We show that in metastatic renal cancer, functionally important metastasis genes are activated via co-option of gene regulatory enhancer modules from distant developmental lineages, thus providing clues to the origins of metastatic cancer. Cancer Discov; 8(7); 850-65. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Epigenetic determinants of metastasis.

Genetic analyses of cancer progression in patient samples and model systems have thus far failed to identify specific mutational drivers of metastasis. Yet, at least in experimental systems, metastatic cancer clones display stable traits that can facilitate progression through the many steps of metastasis. How cancer cells establish and maintain the transcriptional programmes required for metastasis remains mostly unknown. Emerging evidence suggests that metastatic traits may arise from epigenetically altered transcriptional output of the oncogenic signals that drive tumour initiation and early progression. Molecular dissection of such mechanisms remains a central challenge for a comprehensive understanding of the origins of metastasis.

Tumor necrosis factor receptor 2-signaling in CD133-expressing cells in renal clear cell carcinoma.

Compared to normal kidney, renal clear cell carcinomas (ccRCC) contain increased numbers of interstitial, non-hematopoietic CD133+cells that express stem cell markers and exhibit low rates of proliferation. These cells fail to form tumors upon transplantation but support tumor formation by differentiated malignant cells. We hypothesized that killing of ccRCC CD133+ (RCCCD133+) cells by cytotoxic agents might be enhanced by inducing them to divide. Since tumor necrosis factor-alpha (TNF), signalling through TNFR2, induces proliferation of malignant renal tubular epithelial cells, we investigated whether TNFR2 might similarly affect RCCCD133+cells. We compared treating organ cultures of ccRCC vs adjacent nontumour kidney (NK) and RCCCD133+vs NK CD133+ (NKCD133+) cell cultures with wild-type TNF (wtTNF) or TNF muteins selective for TNFR1 (R1TNF) or TNFR2 (R2TNF). In organ cultures, R2TNF increased expression of TNFR2 and promoted cell cycle entry of both RCCCD133+ and NKCD133+ but effects were greater in RCCCD133+. In contrast, R1TNF increased TNFR1 expression and promoted cell death. Importantly, cyclophosphamide triggered much more cell death in RCCCD133+ and NKCD133+cells pre-treated with R2TNF as compared to untreated controls. We conclude that selective engagement of TNFR2 by TNF can drives RCCCD133+ proliferation and thereby increase sensitivity to cell cycle-dependent cytotoxicity.

A hypoxic ticket to the bone metastatic niche.

Hypoxia is a well-characterized driver of aggressive cancer phenotypes, including metastasis. Accumulating evidence suggests that, in addition to having local effects, the consequences of tumour hypoxia can be systemic, leading to the formation of pre-metastatic niches that can later foster metastatic colonization in distant organs. Recent findings have demonstrated that such niches can also form in the bone, possibly revealing new avenues for therapeutic intervention.

Metastatic Competence Can Emerge with Selection of Preexisting Oncogenic Alleles without a Need of New Mutations.

Several experimental models faithfully recapitulate many important facets of human metastatic disease. Here, we have performed whole-exome sequencing in five widely used experimental metastasis models that were independently derived through in vivo selection from heterogeneous human cancer cell lines. In addition to providing an important characterization of these model systems, our study examines the genetic evolution of metastatic phenotypes. We found that in vivo selected highly metastatic cell populations showed little genetic divergence from the corresponding parental population. However, selection of genetic variations that preexisted in parental populations, including the well-established oncogenic mutations KRAS(G13D) and BRAF(G464V), was associated with increased metastatic capability. Conversely, expression of the wild-type BRAF allele in metastatic cells inhibited metastatic outgrowth as well as tumor initiation in mice. Our findings establish that metastatic competence can arise from heterogeneous cancer cell populations without the need for acquisition of additional mutations and that such competence can benefit from further selection of tumor-initiating mutations that seed primary tumorigenesis.

Therapy-induced tumour secretomes promote resistance and tumour progression.

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.