Flower isoforms promote competitive growth in cancer.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system1,2. For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing3, prevent developmental malformations3,4 and replace old tissues during regeneration5. At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins3,4,6. Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated6. However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed4,6,7. Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease8-17. However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage18. This would be undesirable for humans because it might make tumours more aggressive19-21. It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.

Spatial N-glycomics of the normal breast microenvironment reveals fucosylated and high-mannose N-glycan signatures related to BI-RADS density and ancestry.

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.

In vivo modeling of metastatic human high-grade serous ovarian cancer in mice.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

Cell competition and tumor heterogeneity.

Cancers exhibit a remarkable degree of intratumoral heterogeneity (ITH), which results from complex cellular interactions amongst various cell types. This phenomenon provides an opportunity for clonal selection and growth advantages to aggressive cancer cell types, resulting in worse prognosis and challenges to anti-cancer therapy. Cell competition is a conserved mechanism operational in cellular and organ systems, which allows neighboring cells to compare their relative fitness levels and results in the elimination of viable but suboptimal cells. By abuse of this conserved homeostasis mechanism, aggressive cancer cell types gain an advantage over normal cell types by achieving traits like increased proliferation, de-differentiation, and stemness. This review presents recent evidence that cell competition mechanisms actively participate in the regulation of intratumoral cell-cell interactions and thus contribute to ITH, and this process is essential for cancer development and progression.

Building a virtual summer research experience in cancer for high school and early undergraduate students: lessons from the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic posed a unique challenge for summer research programs in 2020, particularly for programs aimed at hands-on experience for younger trainees. The Indiana University Melvin and Bren Simon Comprehensive Cancer Center supports two pipeline programs, which traditionally immerse high school juniors, seniors, and early undergraduate students from underrepresented populations in science in hands-on projects in cancer biology labs. However, due to social distancing policies during the pandemic and reduction of research operations, these students were not physically allowed on campus. Thus, the authors set out to strategically pivot to a wholly virtual curriculum and evaluate the Virtual Summer Research Experience in Cancer outcomes. METHODS: The virtual program included four components: 1. a core science and professional development curriculum led by high school teachers and senior undergraduates; 2. faculty-delivered didactic sessions on cancer science; 3. mentored, virtual research projects with research faculty; and 4. online networking events to encourage vertical mentoring. Outcomes data were measured using a locally created 11-item Research Preparation Scale, daily electronic feedback, and weekly structured evaluation and feedback via Zoom. RESULTS: Outcome data suggested high self-reported satisfaction with the virtual program. Outcome data also revealed the importance of coordination between multiple entities for seamless program implementation. This includes the active recruitment and participation of high school teachers and further investment in information technology capabilities of institutions. CONCLUSIONS: Findings reveal a path to educate and train high school and early undergraduate students in cancer research when hands-on, in-person training is not feasible. Virtual research experiences are not only useful to engage students during public health crises but can provide an avenue for cancer centers to expand their cancer education footprints to remotely located schools and universities with limited resources to provide such experiences to their students.

Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis.

BACKGROUND: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. METHODS: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. RESULTS: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. CONCLUSION: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

Mutational landscape of RNA-binding proteins in human cancers.

RNA Binding Proteins (RBPs) are a class of post-transcriptional regulatory molecules which are increasingly documented to be dysfunctional in cancer genomes. However, our current understanding of these alterations is limited. Here, we delineate the mutational landscape of ∼1300 RBPs in ∼6000 cancer genomes. Our analysis revealed that RBPs have an average of ∼3 mutations per Mb across 26 cancer types. We identified 281 RBPs to be enriched for mutations (GEMs) in at least one cancer type. GEM RBPs were found to undergo frequent frameshift and inframe deletions as well as missense, nonsense and silent mutations when compared to those that are not enriched for mutations. Functional analysis of these RBPs revealed the enrichment of pathways associated with apoptosis, splicing and translation. Using the OncodriveFM framework, we also identified more than 200 candidate driver RBPs that were found to accumulate functionally impactful mutations in at least one cancer. Expression levels of 15% of these driver RBPs exhibited significant difference, when transcriptome groups with and without deleterious mutations were compared. Functional interaction network of the driver RBPs revealed the enrichment of spliceosomal machinery, suggesting a plausible mechanism for tumorogenesis while network analysis of the protein interactions between RBPs unambiguously revealed the higher degree, betweenness and closeness centrality for driver RBPs compared to non-drivers. Analysis to reveal cancer-specific Ribonucleoprotein (RNP) mutational hotspots showed extensive rewiring even among common drivers between cancer types. Knockdown experiments on pan-cancer drivers such as SF3B1 and PRPF8 in breast cancer cell lines, revealed cancer subtype specific functions like selective stem cell features, indicating a plausible means for RBPs to mediate cancer-specific phenotypes. Hence, this study would form a foundation to uncover the contribution of the mutational spectrum of RBPs in dysregulating the post-transcriptional regulatory networks in different cancer types.

Statistical modeling for sensitive detection of low-frequency single nucleotide variants.

BACKGROUND: Sensitive detection of low-frequency single nucleotide variants carries great significance in many applications. In cancer genetics research, tumor biopsies are a mixture of normal and tumor cells from various subpopulations due to tumor heterogeneity. Thus the frequencies of somatic variants from a subpopulation tend to be low. Liquid biopsies, which monitor circulating tumor DNA in blood to detect metastatic potential, also face the challenge of detecting low-frequency variants due to the small percentage of the circulating tumor DNA in blood. Moreover, in population genetics research, although pooled sequencing of a large number of individuals is cost-effective, pooling dilutes the signals of variants from any individual. Detection of low frequency variants is difficult and can be cofounded by sequencing artifacts. Existing methods are limited in sensitivity and mainly focus on frequencies around 2 % to 5 %; most fail to consider differential sequencing artifacts. RESULTS: We aimed to push down the frequency detection limit close to the position specific sequencing error rates by modeling the observed erroneous read counts with respect to genomic sequence contexts. 4 distributions suitable for count data modeling (using generalized linear models) were extensively characterized in terms of their goodness-of-fit as well as the performances on real sequencing data benchmarks, which were specifically designed for testing detection of low-frequency variants; two sequencing technologies with significantly different chemistry mechanisms were used to explore systematic errors. We found the zero-inflated negative binomial distribution generalized linear mode is superior to the other models tested, and the advantage is most evident at 0.5 % to 1 % range. This method is also generalizable to different sequencing technologies. Under standard sequencing protocols and depth given in the testing benchmarks, 95.3 % recall and 79.9 % precision for Ion Proton data, 95.6 % recall and 97.0 % precision for Illumina MiSeq data were achieved for SNVs with frequency > = 1 %, while the detection limit is around 0.5 %. CONCLUSIONS: Our method enables sensitive detection of low-frequency single nucleotide variants across different sequencing platforms and will facilitate research and clinical applications such as pooled sequencing, cancer early detection, prognostic assessment, metastatic monitoring, and relapses or acquired resistance identification.

TFAP2C expression in breast cancer: correlation with overall survival beyond 10 years of initial diagnosis.

Recurrence and death in a significant number of patients with ERα-positive breast cancer occurs 10-20 years after diagnosis. Prognostic markers for late events have been more elusive. TFAP2C (AP2γ) regulates the expression of ERα, the ERα pioneer factors FOXA1 and GATA3, and controls ERα-dependent transcription. The purpose of this investigation is to determine the long-term prognostic value of TFAP2C. A tissue microarray (TMA) consisting of breast tumors from 451 patients with median follow-up time of 10.3 years was created and tested for the expression of TFAP2C by immunohistochemistry. Wilcoxon Rank-Sum and Kruskal-Wallis tests were used to determine if TFAP2C H-scores correlate with other tumor markers. Cox proportional hazards regression models were used to determine whether TFAP2C H-scores and other tumor markers were related to overall and disease-free survival in univariate and multivariable models. TFPAC2 overexpression did not impact overall survival during the first 10 years after diagnosis, but was associated with a shorter survival after 10 years (HR 3.40, 95 % CI 1.58, 7.30; p value = 0.002). This late divergence persisted in ER-positive (HR 2.86, 95 % CI 1.29, 6.36; p value = 0.01) and endocrine therapy-positive subgroups (HR 4.19, 95 % CI 1.72, 10.23; p value = 0.002). For the ER+ and endocrine therapy subgroup, the HR was 3.82 (95 % CI 1.53, 9.50; p value = 0.004). TFAP2C H-scores were not correlated with other tumor markers or related to disease-free survival. In this hypothesis-generating study, we show that higher TFAP2C scores correlate with poor overall survival after 10 years of diagnosis in ERα-positive and endocrine therapy-treated subgroups.

Angiopoietin-2 mediates blood-brain barrier impairment and colonization of triple-negative breast cancer cells in brain.

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple-negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood-brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO-1 and claudin-5 tight junction (TJ) protein structures. Angiopoietin-2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang-2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang-2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang-2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang-2 may serve as potential therapeutics for brain metastasis.

PROGgeneV2: enhancements on the existing database.

BACKGROUND: We recently published PROGgene, a tool that can be used to study prognostic implications of genes in various cancers. The first version of the tool had several areas for improvement. In this paper we present some major enhancements we have made on the existing tool in the new version, PROGgeneV2. RESULTS: In PROGgeneV2, we have made several modifications to enhance survival analysis capability of the tool. First, we have increased the repository of public studies catalogued in our tool by almost two folds. We have also added additional functionalities to perform survival analysis in a variety of new ways. Survival analysis can now be performed on a) single genes b) multiple genes as a signature, c) ratio of expression of two genes, and d) curated/published gene signatures in new version. Users can now also adjust the survival analysis models for available covariates. Users can study prognostic implications of entire gene signatures in different cancer types, which are searchable by keywords. Also, unique to our tool, in the new version, users will be able to upload and use their own datasets to perform survival analysis on genes of interest. CONCLUSIONS: We believe, like its predecessor, PROGGeneV2 will continue to be useful for the scientific community for formulating research hypotheses and designing mechanistic studies. With added datasets PROGgeneV2 is the most comprehensive survival analysis tool available. PROGgeneV2 is available at http://www.compbio.iupui.edu/proggene.

Mutant RAS-driven Secretome Causes Skeletal Muscle Defects in Breast Cancer.

Cancer-induced skeletal muscle defects differ in severity between individuals with the same cancer type. Cancer subtype-specific genomic aberrations are suggested to mediate these differences, but experimental validation studies are very limited. We utilized three different breast cancer patient-derived xenograft (PDX) models to correlate cancer subtype with skeletal muscle defects. PDXs were derived from brain metastasis of triple-negative breast cancer (TNBC), estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) primary breast cancer from a BRCA2-mutation carrier, and pleural effusion from an ER+/PR- breast cancer. While impaired skeletal muscle function as measured through rotarod performance and reduced levels of circulating and/or skeletal muscle miR-486 were common across all three PDXs, only TNBC-derived PDX activated phospho-p38 in skeletal muscle. To further extend these results, we generated transformed variants of human primary breast epithelial cells from healthy donors using HRASG12V or PIK3CAH1047R mutant oncogenes. Mutations in RAS oncogene or its modulators are found in approximately 37% of metastatic breast cancers, which is often associated with skeletal muscle defects. Although cells transformed with both oncogenes generated adenocarcinomas in NSG mice, only HRASG12V-derived tumors caused skeletal muscle defects affecting rotarod performance, skeletal muscle contraction force, and miR-486, Pax7, pAKT, and p53 levels in skeletal muscle. Circulating levels of the chemokine CXCL1 were elevated only in animals with tumors containing HRASG12V mutation. Because RAS pathway aberrations are found in 19% of cancers, evaluating skeletal muscle defects in the context of genomic aberrations in cancers, particularly RAS pathway mutations, may accelerate development of therapeutic modalities to overcome cancer-induced systemic effects. SIGNIFICANCE: Mutant RAS- and PIK3CA-driven breast cancers distinctly affect the function of skeletal muscle. Therefore, research and therapeutic targeting of cancer-induced systemic effects need to take aberrant cancer genome into consideration.

Small Molecule Functional Converter of B-Cell Lymphoma-2 (Bcl-2) Suppresses Breast Cancer Lung Metastasis.

The B-cell lymphoma-2 (Bcl-2) family of proteins plays a vital role in tumorigenesis. Cancer cells utilize the expression of Bcl-2 to evade therapy and develop resistance. Bcl-2 overexpression also causes cancer cells to be more invasive and metastatic. About 80% of cancer deaths are due to metastases, and yet targeted therapies for metastatic cancers are scarce. We discovered a small molecule, BFC1103, which changes the conformation of Bcl-2 to convert the antiapoptotic protein to a proapoptotic protein. BFC1103-induced apoptosis is dependent on the expression levels of Bcl-2, with higher levels causing more apoptosis. BFC1103 suppressed the growth of breast cancer lung metastasis. BFC1103 has the potential for further optimization and development for clinical testing in metastatic cancers that express Bcl-2. This study demonstrates a new approach to target Bcl-2 using a small molecule, BFC1103, to suppress metastatic disease.

Identification and Characterization of a Small Molecule Bcl-2 Functional Converter.

Cancer cells exploit the expression of anti-apoptotic protein Bcl-2 to evade apoptosis and develop resistance to therapeutics. High levels of Bcl-2 leads to sequestration of pro-apoptotic proteins causing the apoptotic machinery to halt. In this study, we report discovery of a small molecule, BFC1108 (5-chloro-N-(2-ethoxyphenyl)-2-[(4-methoxybenzyol)amino]benzamide), which targets Bcl-2 and converts it into a pro-apoptotic protein. The apoptotic effect of BFC1108 is not inhibited, but rather potentiated, by Bcl-2 overexpression. BFC1108 induces a conformational change in Bcl-2, resulting in the exposure of its BH3 domain both in vitro and in vivo. BFC1108 suppresses the growth of triple-negative breast cancer xenografts with high Bcl-2 expression and inhibits breast cancer lung metastasis. This study demonstrates a novel approach to targeting Bcl-2 using BFC1108, a small molecule Bcl-2 functional converter that effectively induces apoptosis in Bcl-2-expressing cancers. SIGNIFICANCE: We report the identification of a small molecule that exposes the Bcl-2 killer conformation and induces death in Bcl-2-expressing cancer cells. Selective targeting of Bcl-2 and elimination of cancer cells expressing Bcl-2 opens up new therapeutic avenues.

Oxygen tension-dependent variability in the cancer cell kinome impacts signaling pathways and response to targeted therapies.

Most cells in solid tumors are exposed to oxygen levels between 0.5% and 5%. We developed an approach that allows collection, processing, and evaluation of cancer and non-cancer cells under physioxia, while preventing exposure to ambient air. This aided comparison of baseline and drug-induced changes in signaling pathways under physioxia and ambient oxygen. Using tumor cells from transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen-dependent differences in cell preference for epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor beta (PDGFRß) signaling. Physioxia caused PDGFRß-mediated activation of AKT and extracellular regulated kinase (ERK) that reduced sensitivity to EGFR and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) inhibition and maintained PDGFRß+ epithelial-mesenchymal hybrid cells with potential cancer stem cell (CSC) properties. Cells in ambient air displayed differential EGFR activation and were more sensitive to targeted therapies. Our data emphasize the importance of oxygen considerations in preclinical cancer research to identify effective drug targets and develop combination therapy regimens.

Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene.

Single-cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single-cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single-cell atlas of mutation carriers with our previously described single-cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal (basal-myoepithelial), luminal progenitor (luminal adaptive secretory precursor, LASP), and mature luminal (luminal hormone sensing) cells in breast tissues. A unique subcluster of cells within LASP cells is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared with non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NFκB, LARP1, and MYC signaling. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin A signaling. BRCA2 mutations were associated with upregulation of IL6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation. SIGNIFICANCE: This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.

Breast-cancer stem cells-beyond semantics.

Intratumoral heterogeneity in breast cancer is well documented. Although the mechanisms leading to this heterogeneity are not understood, a subpopulation of cancer cells, cancer stem cells (CSCs), that have some phenotypic similarities with adult tissue stem cells, has been suggested to contribute to tumour heterogeneity. It has been postulated that these CSCs are dormant, and by virtue of their low proliferative activity and ability to exclude intracellular toxins, are resistant to chemotherapy and radiation therapy. These cells were initially isolated based on the presence of markers such as CD44, CD24, and ALDH1, with further characterisation using mammosphere assay and transplantation into immunodeficient mice. The CSC hypothesis raises several theoretical and practical questions. Does cancer arise in normal mammary stem cells or do some malignant cells acquire a CSC phenotype through clonal evolution? Are CSCs in different molecular (intrinsic) subtypes of breast cancer similar, or do they have distinct properties based on the subtype? Does the CSC phenotype reflect plasticity or the dynamic nature of a few cancer cells? How do these cells acquire invasive behaviour, as they go through epithelial-to-mesenchymal transition and then revert to epithelial phenotype at sites of metastasis in response to tumour microenvironmental and metastasis site-specific cues? It is increasingly recognised that the methods and assays used for identifying CSCs have substantial limitations; does this negate the entire concept? In this Personal View, we argue that the CSC phenotype represents an aggressive clone that survives in an adverse environment through constant evolution and integration of various hallmarks of cancer. This evolution could involve acquiring mutations that permit asymmetric and symmetric division, converting the host immune attack to its own advantage, and plasticity to adapt to sites of metastasis through reversible change in adhesion molecules. We also argue that the cell-type origin of cancer could affect the rate at which CSCs develop in a tumour, with an eventual effect on disease outcome.

A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle.

Skeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.

Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue.

Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays in vitro. Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using in silico prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth in vivo. Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.

Stromal heterogeneity may explain increased incidence of metaplastic breast cancer in women of African descent.

The biologic basis of genetic ancestry-dependent variability in disease incidence and outcome is just beginning to be explored. We recently reported enrichment of a population of ZEB1-expressing cells located adjacent to ductal epithelial cells in normal breasts of women of African ancestry compared to those of European ancestry. In this study, we demonstrate that these cells have properties of fibroadipogenic/mesenchymal stromal cells that express PROCR and PDGFRα and transdifferentiate into adipogenic and osteogenic lineages. PROCR + /ZEB1 + /PDGFRα+ (PZP) cells are enriched in normal breast tissues of women of African compared to European ancestry. PZP: epithelial cell communication results in luminal epithelial cells acquiring basal cell characteristics and IL-6-dependent increase in STAT3 phosphorylation. Furthermore, level of phospho-STAT3 is higher in normal and cancerous breast tissues of women of African ancestry. PZP cells transformed with HRasG12V ± SV40-T/t antigens generate metaplastic carcinoma suggesting that these cells are one of the cells-of-origin of metaplastic breast cancers.