Clonal dominance defines metastatic dissemination in pancreatic cancer.

Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.

Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Pre-existing Functional Heterogeneity of Tumorigenic Compartment as the Origin of Chemoresistance in Pancreatic Tumors.

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.

p53 Is a Master Regulator of Proteostasis in SMARCB1-Deficient Malignant Rhabdoid Tumors.

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRTs) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and ER stress response via a genetically intact MYC-p19ARF-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide a rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRTs.

Stability of an aggregation-prone partially folded state of human profilin-1 correlates with aggregation propensity.

A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of familial forms of ALS (fALS), also known as Lou Gehrig's disease. The pathogenic potential of these mutations is linked to the formation of intracellular inclusions of the mutant proteins and correlates with the mutation-induced destabilization of its native, fully folded state. However, the mechanism by which these mutations promote misfolding and self-assembly is yet unclear. Here, using temperature-jump and stopped-flow kinetic measurements, we show that, during refolding, WT profilin-1 transiently populates a partially folded (PF) state endowed with hydrophobic clusters exposed to the solvent and with no detectable secondary structure. We observed that this conformational state is marginally stable at neutral pH but becomes significantly populated at mildly acidic pH. Interestingly, the fALS-associated mutations did not cause a change in the refolding mechanism of profilin-1, but induced a stabilization of the PF state. In the presence of preformed profilin-1 aggregates, the PF state, unlike the unfolded and folded states, could interact with these aggregates via nonspecific hydrophobic interactions and also increase thioflavin-T fluorescence, revealing its amyloidogenic potential. Moreover, in the variants tested, we found a correlation between conformational stability of PF and aggregation propensity, defining this conformational state as an aggregation-prone folding intermediate. In conclusion, our findings indicate that mutation-induced stabilization of a partially folded state can enhance profilin-1 aggregation and thereby contribute to the pathogenicity of the mutations.

Biophysical analysis of three novel profilin-1 variants associated with amyotrophic lateral sclerosis indicates a correlation between their aggregation propensity and the structural features of their globular state.

Profilin-1 is a small protein involved in actin-mediated cytoskeleton rearrangement. Recently, mutations of profilin-1 have been associated with familial amyotrophic lateral sclerosis. It was previously reported that pathogenic mutations of profilin-1 increase the aggregation propensity of this protein, leaving its function unaffected. However, it is not clear if the mutations act by decreasing the conformational stability or by promoting structural perturbations of the folded state of this protein. In this work we have purified three novel profilin-1 mutants that were recently discovered and have investigated their conformational stability, structural features and aggregation behaviour in vitro. Analysis of the data obtained with the three novel variants, and a global statistical analysis with all profilin-1 mutants so far characterised, indicate significant correlations between aggregation propensity and structural perturbations of the folded state, rather than its conformational stability, in this group of mutants.

The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.

Mutations of Profilin-1 Associated with Amyotrophic Lateral Sclerosis Promote Aggregation Due to Structural Changes of Its Native State.

The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular ß-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.

Packaging signals in the 5'-ends of influenza virus PA, PB1, and PB2 genes as potential targets to develop nucleic-acid based antiviral molecules.

In a previous study a 15-mer phosphorothioate oligonucleotide (S-ON) derived from the packaging signal in the 5' end of segment 1 (PB2) of influenza A virus (designated 5-15b) proved markedly inhibitory to virus replication. Here we investigated whether analogous inhibitory S-ONs targeting the 5' end of segments 2 (PB1) and 3 (PA) could be identified and whether viral resistance to S-ONs can be developed. Similar to our earlier result, 20-mer S-ONs reproducing the 5' ends of segments 2 or 3 (complementary to the 3'-coding regions of PB1 and PA, respectively) exerted a powerful antiviral activity against a variety of influenza A virus subtypes in MDCK cells. Serial passage of the A/Taiwan/1/86 H1N1 strain in the presence of S-ON 5-15b or its antisense as5-15b analogue showed that mutant viruses with reduced susceptibility to the S-ON could indeed be generated, although the resistant viruses displayed reduced replicative fitness. Sequencing the resistant viruses identified mutations in the PB1, PB2, PA and M1 genes. Introduction of these changes into the A/PR/8/34 H1N1 strain by reverse genetics, suggested that alterations to RNA function in the packaging regions of segments 2 and 3 were important in developing resistance to S-ON inhibition. However, many of the other sequence changes induced by S-ON treatment were markedly deleterious to virus fitness. We conclude that packaging signals in the influenza A virus polymerase segments provide feasible targets for nucleic acid-based antivirals that may be difficult for the virus to evade through resistance mutations.