The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs; >200 nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release ...extracellular vesicles (EVs) containing distinct proteomes, microRNAs (miRNAs), and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs, including antisense transcripts and transcripts derived from pseudogenes, are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9-based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.
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•Differential enrichment of coding and noncoding RNAs in EVs suggests regulated export•The majority of mRNAs in EVs are not full length•Smaller mRNAs (∼1 kb) can be transferred from donor to recipient cells by EVs•lncRNAs can be transferred to recipient cells and trafficked to function in the nucleus
Extracellular vesicles (EVs) contain protein and RNA cargo that can be transferred between cells. Hinger et al. identify distinct subsets of cellular coding and long noncoding RNAs that are enriched in EVs that can be functionally transferred between cells, supporting a regulated form of cell-cell communication.
The majority of pancreatic ductal adenocarcinomas (PDAC) are diagnosed late so that surgery is rarely curative. Earlier detection could significantly increase the likelihood of successful treatment ...and improve survival. The aim of the study was to provide proof of principle that point mutations in key cancer genes can be identified by sequencing circulating free DNA (cfDNA) and that this could be used to detect early PDACs and potentially, premalignant lesions, to help target early effective treatment. Targeted next generation sequencing (tNGS) analysis of mutation hotspots in 50 cancer genes was conducted in 26 patients with PDAC, 14 patients with chronic pancreatitis (CP) and 12 healthy controls with
status validated by digital droplet PCR. A higher median level of total cfDNA was observed in patients with PDAC (585 ng/ml) compared to either patients with CP (300 ng/ml) or healthy controls (175 ng/ml). PDAC tissue showed wide mutational heterogeneity, whereas
was the most commonly mutated gene in cfDNA of patients with PDAC and was significantly associated with a poor disease specific survival (p=0.018). This study demonstrates that tNGS of cfDNA is feasible to characterise the circulating genomic profile in PDAC and that driver mutations in
have prognostic value but cannot currently be used to detect early emergence of disease. Importantly, monitoring total cfDNA levels may have utility in individuals "at risk" and warrants further investigation.
Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We ...overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.
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•Large numbers of fluorescent biosensors can be concurrently tracked in barcoded cells•Biosensor activities are synchronized in mixed populations of barcoded cells•Deep learning models facilitate image analysis for biosensor barcoding•Simultaneous biosensor tracking reveals signaling network structures and interactions
Genetically encoded barcodes that uniquely identify cells expressing a particular fluorescent biosensor enable the simultaneous use of a broader set of biosensors in mixed cell populations and dynamic imaging of many cellular responses in a single experiment to reduce inter-experimental variability when interrogating complex biological interactions, such as the downstream consequences of receptor tyrosine kinase activation, with the help of image-based deep learning models for automated barcode unmixing.
The development of the mouse salivary gland involves a tip-driven process of branching morphogenesis that takes place in concert with differentiation into acinar, myoepithelial, and ductal (basal and ...luminal) sub-lineages. By combining clonal lineage tracing with a three-dimensional (3D) reconstruction of the branched epithelial network and single-cell RNA-seq analysis, we show that in tips, a heterogeneous population of renewing progenitors transition from a Krt14+ multipotent state to unipotent states via two transcriptionally distinct bipotent states, one restricted to the Krt14+ basal and myoepithelial lineage and the other to the Krt8+ acinar and luminal lineage. Using genetic perturbations, we show how the differential expression of Notch signaling correlates with spatial segregation, exits from multipotency, and promotes the Krt8+ lineage, whereas Kras activation promotes proacinar fate. These findings provide a mechanistic basis for how positional cues within growing tips regulate the process of lineage segregation and ductal patterning.
mutations drive tumorigenesis by altering cell signaling and the tumor immune microenvironment. Recent studies have shown promise for KRAS-G12C covalent inhibitors, which are advancing rapidly ...through clinical trials. The sequencing and combination of these agents with other therapies including immune checkpoint blockade (ICB) will benefit from strategies that also address the immune microenvironment to improve durability of response.
This paper reviews KRAS signaling and discusses downstream effects on cytokine production and the tumor immune microenvironment. RAS targeted therapy is introduced and perspectives on therapeutic targeting of KRAS-G12C and its immunosuppressive tumor microenvironment are offered.
The availability of KRAS-G12C covalent inhibitors raises hopes for targeting this pervasive oncogene and designing better therapeutic combinations to promote anti-tumor immunity. A comprehensive mechanistic understanding of KRAS immunosuppression is required in order to prioritize agents for clinical trials.
Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial ...fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.
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•Drp1 is required for oncogenic KRas-driven transformation•Drp1 promotes KRas-driven glycolysis•Loss of Drp1 inhibits pancreatic tumorigenesis•Loss of Drp1 impairs mitochondrial metabolism in tumor cells
Nagdas et al. find that the mitochondrial fission GTPase Drp1 is required for KRas-driven transformation and pancreatic tumor growth. The inhibition of Drp1 in cells expressing oncogenic KRas leads to impaired glycolytic flux and the eventual loss of mitochondrial metabolic function.
•No technical bias between fresh frozen and FFPE samples.•A high fraction of adenocarcinoma patients with activating KRAS mutations.•Mutations in TP53, STK11 and SMARCA4 linked to poor prognosis in ...adenocarcinoma.•Mutations in CSMD3 linked to better prognosis in squamous cell carcinoma.•Co-mutations in TP53 or STK11 confer poor prognosis in KRAS positive patients.
Non-small cell lung cancer (NSCLC) is a heterogeneous disease with unique combinations of somatic molecular alterations in individual patients, as well as significant differences in populations across the world with regard to mutation spectra and mutation frequencies. Here we aim to describe mutational patterns and linked clinical parameters in a population-based NSCLC cohort.
Using targeted resequencing the mutational status of 82 genes was evaluated in a consecutive Swedish surgical NSCLC cohort, consisting of 352 patient samples from either fresh frozen or formalin fixed paraffin embedded (FFPE) tissues. The panel covers all exons of the 82 genes and utilizes reduced target fragment length and two-strand capture making it compatible with degraded FFPE samples.
We obtained a uniform sequencing coverage and mutation load across the fresh frozen and FFPE samples by adaption of sequencing depth and bioinformatic pipeline, thereby avoiding a technical bias between these two sample types. At large, the mutation frequencies resembled the frequencies seen in other western populations, except for a high frequency of KRAS hotspot mutations (43%) in adenocarcinoma patients. Worse overall survival was observed for adenocarcinoma patients with a mutation in either TP53, STK11 or SMARCA4. In the adenocarcinoma KRAS-mutated group poor survival appeared to be linked to concomitant TP53 or STK11 mutations, and not to KRAS mutation as a single aberration. Similar results were seen in the analysis of publicly available data from the cBioPortal. In squamous cell carcinoma a worse prognosis could be observed for patients with MLL2 mutations, while CSMD3 mutations were linked to a better prognosis.
Here we have evaluated the mutational status of a NSCLC cohort. We could not confirm any survival impact of isolated driver mutations. Instead, concurrent mutations in TP53 and STK11 were shown to confer poor survival in the KRAS-positive adenocarcinoma subgroup.
Although endometriosis is a benign disease, it is associated with cancer-related gene mutations, such as
or
. Endometriosis is associated with elevated levels of inflammatory factors that cause ...severe pain. In a previous study, we demonstrated that
or
mutations are associated with the activation of cell proliferation, migration, and invasion in a patient-derived immortalized endometriotic cell line, HMOsisEC10. In this study, we investigated the effects of these mutations on progesterone resistance. Since the HMOsisEC10 had suppressed progesterone receptor (PR) expression, we transduced PR-B to HMOsisEc10 cell lines including
mutant and
mutant cell lines. We conducted a migration assay, invasion assay, and MTT assay using dienogest and medroxyprogestrone acetate. All cell lines showed progesterone sensitivity with or without mutations. Regarding inflammatory factors, real-time quantitative RT-PCR revealed that the
mutation cell line exhibited no suppression of Cox-2 and mPGES-1 on progesterone treatment, whereas IL-6, MCP-1, VEGF, and CYP19A1 were significantly suppressed by progesterone in both mutated cell lines. Our results suggest that
mutation and
mutation in endometriotic cells may not be associated with progesterone resistance in terms of aggressiveness. However,
mutations may be associated with progesterone resistance in the context of pain.
Pulmonary invasive mucinous adenocarcinomas (IMAs) often present with spatially separate lung lesions. Clonal relationship between such lesions, particularly those involving contralateral lobes, is ...not well established. Here, we used comparative genomic profiling to address this question.
Patients with genomic analysis performed on two IMAs located in different lung regions were identified. Molecular assays included DNA-based next-generation sequencing (NGS) for 410 to 468 genes (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets), RNA-based NGS for 62 genes (Memorial Sloan Kettering-Fusion), or non-NGS assays.
Comparative genomic profiling was performed on two separate IMAs in 24 patients, of whom 19 had contralateral lesions. Tumors from all but one patient shared matching driver alterations, including KRAS (n = 19), NRG1 (n = 2), ERBB2 (n = 1) or BRAF (n = 1). In addition, in patients with paired tumors profiled by NGS (n = 12), shared driver alterations were accompanied by up to 4 (average 2.6) other identical mutations, further supporting the clonal relationship between the tumors. Only in a single patient separate IMAs harbored entirely nonoverlapping mutation profiles, supporting clonally unrelated, distinct primary tumors. Notably, in a subset of patients (n = 3), molecular testing confirmed a clonal relationship between the original resected IMAs and subsequent contralateral IMA presenting after an extremely long latency (8.1–11.7 y).
Comparative molecular profiling supports that nearly all separate pulmonary IMA lesions represent intrapulmonary spread arising from a single tumor and documents a subset with a remarkably protracted course of intrapulmonary progression. This study reinforces the unique biology and clinical behavior of IMAs while further highlighting the value of genomic testing for clarifying the clonal relationship between multiple lung carcinomas.
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