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1. Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

   https://doi.org/10.1186/s12885-022-10311-1  

   Lo, April ; McSharry, Maria ; Berger, Alice H. ( December 2022 , BMC Cancer)

   Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.

   We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.

   Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRAS^G12Vand KRAS^Q61Hcells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45^M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.

   Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

    

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2. miR-489 Confines Uncontrolled Estrogen Signaling through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer

   https://doi.org/10.3390/ijms23158086  

   Soni, Mithil ; Saatci, Ozge ; Gupta, Gourab ; Patel, Yogin ; Keerthi Raja, Manikanda Raja ; Li, Jie ; Liu, Xinfeng ; Xu, Peisheng ; Wang, Hongjun ; Fan, Daping ; et al ( August 2022 , International Journal of Molecular Sciences)

   Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor-positive tumors and are associated with a better prognosis due to response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen-resistant cell lines suggested the potential role of miR-489 in the regulation of estrogen signaling and development of tamoxifen resistance. Our in vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance, while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen-regulated miRNA that negatively regulates estrogen receptor signaling by using at least the following two mechanisms: (i) modulation of the ER phosphorylation status by inhibiting MAPK and AKT kinase activities; (ii) regulation of nuclear-to-cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 can break the positive feed-forward loop between the estrogen-Erα axis and p38 MAPK in breast cancer cells, which is necessary for its function as a transcription factor. Overall, our study unveiled the underlying molecular mechanism by which miR-489 regulates an estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers. 

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3. Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics

   https://doi.org/10.1016/j.cels.2023.05.003  

   Morse, David B. ; Michalowski, Aleksandra M. ; Ceribelli, Michele ; De Jonghe, Joachim ; Vias, Maria ; Riley, Deanna ; Davies-Hill, Theresa ; Voss, Ty ; Pittaluga, Stefania ; Muus, Christoph ; et al ( June 2023 , Cell Systems)

   Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluores- cent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors. 

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4. Microfluidic device for expedited tumor growth towards drug evaluation

   https://doi.org/10.1039/C8LC01250D  

   Uhl, Christopher George ; Liu, Yaling ( April 2019 , Lab on a Chip)

   Patient derived organoids have emerged as robust preclinical models for screening anti-cancer therapeutics. Current 2D culturing methods do not provide physiological responses to therapeutics, therefore 3D models are being developed to better reproduce physiological responses. 3D culturing however often requires large initial cell populations and one week to one month to grow tumors ready for therapeutic testing. As a solution a 3D culturing system has been developed capable of producing physiologically relevant tumors in an expedited fashion while only requiring a small number of initial cancer cells. A bi-layer microfluidic system capable of facilitating active convective nutrient supply to populations of cancer cells facilitates expedited growth of cancer cells when starting with populations as small as 8 cells. The system has been shown to function well with adherent and non-adherent cell types by expediting cell growth by a factor ranging from 1.27 to 4.76 greater than growth under static conditions. Utilizing such an approach has enable to formation of tumors ready for therapeutic screening within 3 days and the ability to perform therapeutic screening within the microfluidic system is demonstrated. A mathematical model has been developed which allows for adjustments to be made to the dynamic delivery of nutrients in order to efficiently use culture media without excessive waste. We believe this work to be the first attempt to grow cancers in an expedited fashion utilizing only a convective nutrient supply within a microfluidic system which also facilitates on-device therapeutic screening. The developed microfluidic system and cancer growth method have the potential to offer improved drug screening for patients in clinical settings. 

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5. Infiltration of CD8 + T cells into tumor cell clusters in triple-negative breast cancer

   https://doi.org/10.1073/pnas.1817652116  

   Li, Xuefei ; Gruosso, Tina ; Zuo, Dongmei ; Omeroglu, Atilla ; Meterissian, Sarkis ; Guiot, Marie-Christine ; Salazar, Adam ; Park, Morag ; Levine, Herbert ( February 2019 , Proceedings of the National Academy of Sciences)

   Infiltration of${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T lymphocytes into solid tumors is associated with good prognosis in various types of cancer, including triple-negative breast cancer (TNBC). However, the mechanisms underlying different infiltration levels are largely unknown. Here, we have characterized the spatial profile of${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T cells around tumor cell clusters (tightly connected tumor cells) in the core and margin regions in TNBC patient samples. We found that in some patients, the${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T cell density first decreases when moving in from the boundary of the tumor cell clusters and then rises again when approaching the center. To explain various infiltration profiles, we modeled the dynamics of T cell density via partial differential equations. We spatially modulated the diffusion/chemotactic coefficients of T cells (to mimic physical barriers) or introduced the localized secretion of a diffusing T cell chemorepellent. Combining the spatial-profile analysis and the modeling led to support for the second idea; i.e., there exists a possible chemorepellent inside tumor cell clusters, which prevents${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T cells from infiltrating into tumor cell clusters. This conclusion was consistent with an investigation into the properties of collagen fibers which suggested that variations in desmoplastic elements does not limit infiltration of${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T lymphocytes, as we did not observe significant correlations between the level of T cell infiltration and fiber properties. Our work provides evidence that${\mathrm{C}\mathrm{D}\mathrm{8}}^{+}$T cells can cross typical fibrotic barriers and thus their infiltration into tumor clusters is governed by other mechanisms possibly involving a local repellent.

    

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