RNA sequencing (RNA-seq) is a gold standard technology for studying gene and transcript expression. Different transcripts from the same gene are usually determined by varying combinations of exons within the gene, formed by splicing events. One method of studying differential alternative splicing between groups in short-read RNA-seq experiments is through differential exon usage (DEU) analysis, which uses exon-level read counts along with downstream statistical testing strategies. However, the standard exon counting method does not consider exon-junction information, which may reduce the statistical power in detecting splicing alterations. We present a new workflow for differential splicing analysis, called differential exon-junction usage (DEJU). This DEJU analysis workflow adopts a new feature quantification approach that jointly summarises exon and exon–exon junction reads, which are then integrated into the established Rsubread-edgeR/limma frameworks. We performed comprehensive simulation studies to benchmark the performance of DEJU against existing methods. We also applied DEJU to a mouse mammary gland RNA-seq dataset, revealing biologically meaningful splicing events that could not be detected previously. We demonstrate that incorporating exon–exon junction reads significantly improves the detection of differential splicing events. The proposed DEJU workflow offers increased statistical power and computational efficiency compared to widely used existing approaches, while effectively controlling the false discovery rate.

Fibroblasts form a major component of the stroma in normal mammary tissue and breast tumors. Here, we have applied longitudinal single-cell transcriptome profiling of >45,000 fibroblasts in the mouse mammary gland across five different developmental stages and during oncogenesis. In the normal gland, diverse stromal populations were resolved, including lobular-like fibroblasts, committed preadipocytes and adipogenesis-regulatory, as well as cycling fibroblasts in puberty and pregnancy. These specialized cell types appear to emerge from CD34high mesenchymal progenitor cells, accompanied by elevated Hedgehog signaling. During late tumorigenesis, heterogeneous cancer-associated fibroblasts (CAFs) were identified in mouse models of breast cancer, including a population of CD34- myofibroblastic CAFs (myCAFs) that were transcriptionally and phenotypically similar to senescent CAFs. Moreover, Wnt9a was demonstrated to be a regulator of senescence in CD34- myCAFs. These findings reflect a diverse and hierarchically organized stromal compartment in the normal mammary gland that provides a framework to better understand fibroblasts in normal and cancerous states.

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC, which is abnormally highly expressed in ~70% of human cancers, we use a genome-wide CRISPR gene knockout screen in Eµ-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discover that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerates c-MYC-driven lymphoma development in mice. MYC-driven lymphomas lacking GATOR1 display constitutive mTOR pathway activation and are highly sensitive to mTOR inhibitors, both in vitro and in vivo. These findings identify GATOR1 suppression of mTORC1 as a tumor suppressive mechanism in MYC-driven lymphomagenesis and suggest an avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition.

Abstract Minor splicing is an under-appreciated splicing system required for the correct expression of ~700 genes in the human genome. This small subset of genes (0.35%) harbours introns containing non-canonical splicing sequences that are recognised uniquely by the minor spliceosome and cannot be processed by the major spliceosome. Using in vivo zebrafish and mouse cancer models, we show that heterozygous expression of Rnpc3, encoding a unique protein component of the minor spliceosome, restricts the growth and survival of liver, lung and gastric tumours without impacting healthy cells. RNPC3 knockdown in human lung cancer-derived A549 cells also impairs cell proliferation and RNA-seq analysis reveals a robust and selective disruption to minor intron splicing and transcription-wide effects on gene expression. We further demonstrate that these perturbations are accompanied by DNA replication stress, DNA damage, accumulation of TP53 protein and activation of a Tp53-dependent transcriptional program that induces cell cycle arrest and apoptosis. Together our data reveal a vulnerability of cancer cells to minor splicing inhibition that restricts tumour growth.

The release of Ca2+ ions from endoplasmic reticulum calcium stores is a key event in a variety of cellular processes, including gene transcription, migration and proliferation. This release of Ca2+ often occurs through inositol 1,4,5-triphosphate receptors and the activity of these channels and the levels of stored Ca2+ in the endoplasmic reticulum are important regulators of cell death in cancer cells. A recently identified Ca2+ channel of the endoplasmic reticulum is transmembrane and coiled-coil domains 1 (TMCO1). In this study, we link the overexpression of TMCO1 with prognosis in node-positive basal breast cancer patients. We also identify interacting proteins of TMCO1, which include endoplasmic reticulum-resident proteins involved in Ca2+ regulation and proteins directly involved in nucleocytoplasmic transport. Interacting proteins included nuclear transport proteins and TMCO1 was shown to have both nuclear and endoplasmic reticulum localisation in MDA-MB-231 basal breast cancer cells. These studies also define a role for TMCO1 in the regulation of breast cancer cells in their sensitivity to BCL-2/MCL-1 inhibitors, analogous to the role of inositol 1,4,5-triphosphate receptors in the regulation of cell death pathways activated by these agents.

Inheritance of a BRCA2 pathogenic variant conveys a substantial life-time risk of breast cancer. Identification of the cell(s)-of-origin of BRCA2-mutant breast cancer and targetable perturbations that contribute to transformation remains an unmet need for these individuals who frequently undergo prophylactic mastectomy. Using preneoplastic specimens from age-matched, premenopausal females, here we show broad dysregulation across the luminal compartment in BRCA2mut/+ tissue, including expansion of aberrant ERBB3lo luminal progenitor and mature cells, and the presence of atypical oestrogen receptor (ER)-positive lesions. Transcriptional profiling and functional assays revealed perturbed proteostasis and translation in ERBB3lo progenitors in BRCA2mut/+ breast tissue, independent of ageing. Similar molecular perturbations marked tumours bearing BRCA2-truncating mutations. ERBB3lo progenitors could generate both ER+ and ER- cells, potentially serving as cells-of-origin for ER-positive or triple-negative cancers. Short-term treatment with an mTORC1 inhibitor substantially curtailed tumorigenesis in a preclinical model of BRCA2-deficient breast cancer, thus uncovering a potential prevention strategy for BRCA2 mutation carriers.

Differences in shape can be a distinguishing feature between different cell types, but the shape of a cell can also be dynamic. Changes in cell shape are critical when cancer cells escape from the primary tumor and undergo major morphological changes that allow them to squeeze between endothelial cells, enter the vasculature, and metastasize to other areas of the body. A shift from rounded to spindly cellular geometry is a consequence of epithelial-mesenchymal plasticity, which is also associated with changes in gene expression, increased invasiveness, and therapeutic resistance. However, the consequences and functional impacts of cell shape changes and the mechanisms through which they occur are still poorly understood. Here, we demonstrate that altering the morphology of a cell produces a remodeling of calcium influx via the ion channel PIEZO1 and identify PIEZO1 as an inducer of features of epithelial-to-mesenchymal plasticity. Combining automated epifluorescence microscopy and a genetically encoded calcium indicator, we demonstrate that activation of the PIEZO1 force channel with the PIEZO1 agonist, YODA 1, induces features of epithelial-to-mesenchymal plasticity in breast cancer cells. These findings suggest that PIEZO1 is a critical point of convergence between shape-induced changes in cellular signaling and epithelial-mesenchymal plasticity in breast cancer cells. A multidisciplinary approach suggests the importance of cellular geometry in modulating calcium signals in breast cancer cells via the PIEZO1 force sensing channel which is also associated with breast cancer cell plasticity.

Hormone-receptor-positive (HR+) luminal cells largely mediate the response to estrogen and progesterone during mammary gland morphogenesis. However, there remains a lack of consensus on the precise nature of the precursor cells that maintain this essential HR+ lineage. Here we refine the identification of HR+ progenitors and demonstrate their unique regenerative capacity compared to mature HR+ cells. HR+ progenitors proliferate but do not expand, suggesting rapid differentiation. Subcellular resolution, 3D intravital microscopy was performed on terminal end buds (TEBs) during puberty to dissect the contribution of each luminal lineage. Surprisingly, HR+ TEB progenitors were highly elongated and motile compared to columnar HR- progenitors and static, conoid HR+ cells within ducts. This dynamic behavior was also observed in response to hormones. Development of an AI model for motility dynamics analysis highlighted stark behavioral changes in HR+ progenitors as they transitioned to mature cells. This work provides valuable insights into how progenitor behavior contributes to mammary morphogenesis.

Although lineage-specific genes have been identified in the mammary gland, little is known about the contribution of the 3D genome organization to gene regulation in the epithelium. Here, we describe the chromatin landscape of the three major epithelial subsets through integration of long- and short-range chromatin interactions, accessibility, histone modifications, and gene expression. While basal genes display exquisite lineage specificity via distal enhancers, luminal-specific genes show widespread promoter priming in basal cells. Cell specificity in luminal progenitors is largely mediated through extensive chromatin interactions with super-enhancers in gene-body regions in addition to interactions with polycomb silencer elements. Moreover, lineage-specific transcription factors appear to be controlled through cell-specific chromatin interactivity. Finally, chromatin accessibility rather than interactivity emerged as a defining feature of the activation of quiescent basal stem cells. This work provides a comprehensive resource for understanding the role of higher-order chromatin interactions in cell-fate specification and differentiation in the adult mouse mammary gland.

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new tumor driving processes we have used a genome-wide CRISPR/Cas9 knockout screen in Eµ-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo . We uncovered that loss of either of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice, indeed to an extent comparable to loss of p53. In human lymphomas mutations or low expression of the GATOR1 complex genes were mutually exclusive with defects in p53 and correlated with poor survival outcomes for patients with high MYC-expressing cancers. Lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors, both in vitro and in vivo . These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis, and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition.Statement of Significance Our in vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor in c-MYC-driven lymphomagenesis. GATOR1 deficiency obviates the pressure to lose p53 during c-MYC driven lymphoma development and primes these malignant cells for heightened sensitivity to mTOR inhibition, suggesting a novel therapeutic approach.### Competing Interest StatementThe authors have declared no competing interest.

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53(+/-) heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.

BACKGROUND:After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue.METHODS:In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR.RESULTS:Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa.CONCLUSIONS:Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

Minor splicing is a second splicing system required for the correct expression of ∼700 human minor intron-containing genes (MIGs). Many MIGs are expressed in vigorously proliferating cells and are frequently dysregulated in cancer including BRAF, ERK, JNK and p38 . Minor splicing is carried out by the minor spliceosome which comprises several unique components, including a 65kDa protein encoded by RNPC3 . We show that Rnpc3 heterozygosity reduces tumour burden in a broad spectrum of in vivo cancer settings, without harming normal tissues. Using the collective power of zebrafish, mouse and human cancer models, we reveal a sequence of events connecting Rnpc3 deficiency and impaired splicing of MIGs to DNA damage and activation of a Tp53-dependent transcriptional program that restricts tumour burden by inducing cell cycle arrest and apoptosis. Interrogation of human liver and lung cancer transcriptomes curated in TCGA revealed that the expression of many of the genes encoding protein components of the minor spliceosome is upregulated in these cancers. This is accompanied by upregulation of the expression of MIGs that are enriched in cell cycle and DNA damage pathways. These findings suggest that cancer cells can invoke mechanisms to increase the efficiency of minor splicing to support their high proliferation rates. Finally, Kaplan Meier survival analysis shows that highly expressed MIGs are frequently associated with poor patient survival. Taken together, these results indicate that the minor spliceosome offers a therapeutically viable target for the treatment of a broad spectrum of cancers.

Dysregulation in calcium signalling is implicated in several cancer-associated processes, including cell proliferation, migration, invasion and therapy resistance. Modulators of specific calcium-regulating proteins have been proposed as promising future therapeutic agents for some cancers. Alterations in calcium signalling have been extensively studied in some cancers; however, this area of research is highly underexplored in medulloblastoma (MB), the most common paediatric malignant brain tumour. Current MB treatment modalities are not completely effective and can result in several long-lasting mental complications. Hence, new treatment strategies are needed. In this study, we sought to probe the landscape of calcium signalling regulators to uncover those most likely to be involved in MB tumours. We investigated the expression of calcium signalling regulator genes in MB patients using publicly available datasets. We stratified the expression level of these genes with MB molecular subgroups, tumour metastasis and patient survival to uncover correlations with clinical features. Of particular interest was CACNA1 genes, in which we were able to show a developmentally-driven change in expression within the cerebellum, MB's tissue of origin, highlighting a potential influence on tumour incidence. This study lays a platform for future investigations into molecular regulators of calcium signalling in MB formation and progression.

Transient receptor potential cation channel subfamily V (TRPV) channels play important roles in a variety of cellular processes. One example includes the sensory role of TRPV1 that is sensitive to elevated temperatures and acidic environments and is activated by the hot pepper component capsaicin. Another example is the importance of the highly Ca2+ selective channels TRPV5 and TRPV6 in Ca2+ absorption/reabsorption in the intestine and kidney. However, in some cases such as TRPV4 and TRPV6, breast cancer cells appear to overexpress TRPV channels. Moreover, TRPV mediated Ca2+ influx may contribute to enhanced breast cancer cell proliferation and other processes important in tumor progression such as angiogenesis. It appears that the overexpression of some TRPV channels in breast cancer and/or their involvement in breast cancer cell processes, processes important in the tumor microenvironment or pain may make some TRPV channels potential targets for breast cancer therapy. In this review, we provide an overview of TRPV expression in breast cancer subtypes, the roles of TRPV channels in various aspects of breast cancer progression and consider implications for future therapeutic approaches.

Epithelial to mesenchymal transition (EMT) in cancer is important in therapeutic resistance and invasiveness. Calcium signaling is key to the induction of EMT in breast cancer cells. Although inhibition of specific calcium-permeable ion channels regulates the induction of a sub-set of EMT markers in breast cancer cells, it is still unclear if activation of a specific calcium channel can be a driver for the induction of EMT events. In this study, we exploited the availability of a selective pharmacological activator of the calcium-permeable ion channel TRPV4 to assess the direct role of calcium influx in EMT marker induction. Gene association studies revealed a link between TRPV4 and gene-ontologies associated with EMT and poorer relapse-free survival in lymph node-positive basal breast cancers. TRPV4 was an important component of the calcium influx phase induced in MDA-MB-468 breast cancer cells by the EMT inducer epidermal growth factor (EGF). Pharmacological activation of TRPV4 then drove the induction of a variety of EMT markers in breast cancer cells. These studies demonstrate that calcium influx through specific pathways appears to be sufficient to trigger EMT events.

Neuronal calcium sensor‐1 (NCS‐1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS‐1 and breast cancer molecular subtypes and the effects of NCS‐1 silencing on calcium (Ca2+) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS‐1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA‐MB‐231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS‐1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS‐1 silencing suppressed unstimulated basal Ca2+ influx. NCS‐1 silencing in MDA‐MB‐231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS‐1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store‐operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS‐1 in clinical breast cancer samples. This newly identified association between NCS‐1 and basal breast cancers, together with the identification of the role of NCS‐1 in the regulation of the effects of doxorubicin in MDA‐MB‐231 breast cancer cells, suggests that NCS‐1 and/or pathways regulated by NCS‐1 may be important in the treatment of basal breast cancers in women.

The remodeling of specific calcium-permeable ion channels is a feature of some breast cancer subtypes. ORAI1 is a protein that forms a calcium-permeable ion channel responsible for store-operated calcium entry (SOCE) in a variety of cell types. ORAI3, a related isoform, is not a regulator of SOCE in most cell types. However, ORAI3 does control SOCE in many estrogen receptor-positive breast cancer cell lines, where it also controls proliferation. ORAI1 is a well-characterized regulator of the proliferation and migration of many basal breast cancer cells; however, the role of ORAI3 in these types of breast cancer cells remains unclear. Here, we sought to define ORAI1 and ORAI3 expression in breast cancer cell lines of different molecular subtypes and assess the potential role and regulation of ORAI3 in basal breast cancer cells. Our study demonstrates that elevated ORAI1 is a feature of basal-like breast cancers, while elevated ORAI3 is a feature of luminal breast cancers. Intriguingly, we found that ORAI3 is over-expressed in the mesenchymal subtype of triple-negative breast cancer. Given this, we assessed ORAI3 levels in the presence of two inducers of the mesenchymal phenotype, hypoxia and epidermal growth factor (EGF). Hypoxia induced ORAI3 levels in basal breast cancer cell lines through a pathway involving hypoxia-inducible factor-1 alpha (HIF1α. The silencing of ORAI3 attenuated hypoxia-associated phosphorylation of the EGF receptor (EGFR) and the expression of genes associated with cell migration and inflammatory/immune responses in the MDA-MB-468 model of basal breast cancer. Although elevated ORAI3 levels were not associated with survival; basal, estrogen receptor-negative and triple-negative breast cancers with high ORAI3 and low ORAI1 levels were associated with poorer clinical outcomes. This study defines ORAI3 as a potential fine-tuner for processes relevant to the progression of basal breast cancers.

Transient receptor potential cation channel subfamily V (TRPV) channels play important roles in a variety of cellular processes. One example includes the sensory role of TRPV1 that is sensitive to elevated temperatures and acidic environments and is activated by the hot pepper component capsaicin. Another example is the importance of the highly Ca 2+ selective channels TRPV5 and TRPV6 in Ca 2+ absorption/reabsorption in the intestine and kidney. However, in some cases such as TRPV4 and TRPV6, breast cancer cells appear to overexpress TRPV channels. Moreover, TRPV mediated Ca 2+ influx may contribute to enhanced breast cancer cell proliferation and other processes important in tumor progression such as angiogenesis. It appears that the overexpression of some TRPV channels in breast cancer and/or their involvement in breast cancer cell processes, processes important in the tumor microenvironment or pain may make some TRPV channels potential targets for breast cancer therapy. In this review, we provide an overview of TRPV expression in breast cancer subtypes, the roles of TRPV channels in various aspects of breast cancer progression and consider implications for future therapeutic approaches.