Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy

Escaping growth arrest in cancer

We now have a general idea of which genes and cellular pathways are central in cancer development: uncontrolled growth governs establishment and spread of the disease. Nevertheless, to fully appreciate the consequences of the interplay of driver mutations in this genetic disease it is imperative we understand the mechanisms behind uncontrolled growth, or escape of growth arrest, in cancer. It is still not fully understood how cells escape the tumor-suppressing activity of p53, the most mutated gene in cancer. How p53 is regulated and recruited to protect against tumor formation is well known, but it is less well known how it executes this function. By studying the tumor-suppressive activity of p53 in both a biased and unbiased manner I have identified various molecules and mechanisms that may intersect with p53 function when it comes to uncontrolled cell growth, or tumor initiation. I have found that there is a reciprocal regulation of p53 and growth stimulatory signals, since activation of p53 results in an arrest in the cell cycle, or senescence, by blocking growth signaling, and that hyperactive growth factor signaling can override p53 function. Interestingly, plasminogen activator inhibitor-1 (PAI-1), one of the molecules I found to be causally involved in senescence, and which normally is activated by p53 under stressful conditions, is a secreted protein and has its most direct function on the outside of a cell. Hence, it can be regarded a gatekeeper of cell cycle progression. My findings also suggest that a molecule that formerly was coupled to extra-cellular cross-talk between cells and wound-healing apparently directly controls cycling of a cell. PAI-1 was already known to be involved in tumor progression (a process dependent on excessive communication between cells), since it highly influences metastatic behavior of a cell. For example, quantification of this molecule is used in the clinic as a marker for breast cancer progression. Next to this I screened for genes that, when hyperactivated, interfere with anti-tumorigenic p53 activity, and found that lysophosphatidic acid (LPA) activity induce a senescence-bypass. LPA is a lipid involved in growth and wound healing processes. This further supports the notion that growth factor- and p53 signaling are linked. I also found that TGFbeta, a molecule with strong tumor suppressive activity, needs PAI-1 for its anti-tumorigenic effect. One of the biggest questions regarding TGFbeta dependent tumorigenesis is what mutations drive the transition from tumor intitiation to tumor progression or, in other words, what mutations make cells less sensitive to innate anti-tumorigenic activity of stress signals. I hypothesize that PAI-1 may be a central player and necessary downstream of both anti-tumorigenic p53- and TGFbeta signaling. My findings shed light on how intra-cellular tumor-suppressive gene programs result in specific communicative behavior of cells, and support the notion that wound healing and tumor-progression have parallel mechanisms. Furthermore, it provides us insight in the cross-talk between some of the key players in cancer initiation and progression which were previously not linked

Senescence, Wound Healing and Cancer. The PAI-1 Connection

Prolonged propagation of primary diploid fibroblasts in culture activates an ageing process known as replicative senescence, which is considered to provide a barrier against oncogenic transformation. Remarkably, both cell autonomous tumor‑suppressive and cell nonautonomous tumor‑promoting effects of senescent cells have been reported. Recently, we described that the p53 target gene plasminogen activator inhibitor‑1 (PAI‑1) is an essential mediator of replicative senescence. PAI‑1 antagonizes the protease urokinase‑type plasminogen activator (uPA). Both are secreted factors and involved in heterotypic signaling processes such as wound healing, angiogenesis and metastasis. Both uPA and PAI‑1 are expressed in senescent cells and their relative abundance controls proliferation downstream of p53. Here, we present data that the effects of PAI‑1 and uPA in the senescence response are not strictly cell autonomous. We discuss these findings in the context of the emerging roles of PAI‑1 and uPA in heterotypic cellular signaling in senescence, wound healing and metastasis

Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence

genesPAI-1PI(3)KPKBGSK3cyclin D1p19ARFp21CIP1uPAp16INK4Acyclin EPCNAp53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence — a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway1, 2, 3. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K–PKB–GSK3 pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K–PKB–GSK3–cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53

TBX-3, the Gene Mutated in Ulnar-Mammary Syndrome, Is a Negative Regulator of p19ARF and Inhibits Senescence

Prolonged culturing of rodent cells in vitro activates p19ARF (named p14ARF in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19ARF-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar- Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19ARF and human p14ARF. We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19ARF and human p14ARF expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14ARF

TBX-3, the Gene Mutated in Ulnar-Mammary Syndrome, Is a Negative Regulator of p19ARF and Inhibits Senescence

Prolonged culturing of rodent cells in vitro activates p19ARF (named p14ARF in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19ARF-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar- Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19ARF and human p14ARF. We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19ARF and human p14ARF expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14ARF

A MYC-driven change in mitochondrial dynamics limits YAP/TAZ function in mammary epithelial cells and breast cancer

In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors

Bypassing cellular senescence by genetic screening tools

8 páginas, figuras.Bypassing cellular senescence is a prerequisite step in the tumorigenic transformation. It has long been known that loss of a key tumour suppressor gene, such as p53 or pRB, is necessary but not sufficient for spontaneous cellular immortalisation. Therefore, there must be additional mutations and/or epigenetic alterations required for immortalisation to occur. Early work on these processes included somatic-cell genetic studies to estimate the number of senescence genes and nowadays are completed by in vivo models and with the requirements to bypass senescence induced by oncogenic transformation in stem cells. These principal studies laid the foundation for the field of senescence/immortalisation but were labour intensive and the results were somewhat limited. Using retroviral-based functional genetic screening, we and others identified universal genes regulating senescence/immortalisation (either by gain or loss of function) and found that some of these genes are widely altered in human tumours. We also explored the molecular mechanisms throughout these genes that regulate senescence and established the causality of the genetic alteration in tumorigenesis. The identification of genes and pathways regulating senescence/immortalisation could provide novel molecular targets for the treatment and/or prevention of cancer.Peer reviewe