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Breast cancers are categorized into subtypes with distinctive therapeutic vulnerabilities based on expression of clinically targetable receptors and other genes that mimic cell types of the normal gland. Here, we tested the role of the plasma membrane-integral glycoprotein Mcam in breast cancer cell state control and tumorigenicity using a murine tumor cell line (Py230), that exhibits lineage and tumor subtype plasticity. Mcam knockdown (KD) Py230 cells exhibit increased mesenchymal morphology, migration, Src/Fak/Mapk/Paxillin adhesion complex signaling and Pi3K/Akt, Stat3 and Stat5a activation. They also show a transcriptional switch from a hormone-sensing/luminal progenitor state toward alveolar and basal cell states. Reminiscent of archival human breast cancers and patient derived organoid expression data associated with endocrine resistant disease, Mcam KD Py230 cells were refractory to growth inhibition by tamoxifenin vitro. Endocrine resistance and cell state change resulting from Mcam KD were reversed by inhibition of Stat3 or the upstream activating kinase Ck2. Finally, while Mcam KD cells exhibited more aggressive phenotypesin vitro, they showed reduced tumorigenicity and lacked Sox10+/neural crest cell state acquisitionin vivo. Our studies uncover breast cancer cell state plasticity dependent on Mcam, Ck2, and Stat3 with implications for progression, evasion of targeted therapies and combination therapy design.

Supplementary Data from Elevated Poly-(ADP-Ribose)-Polymerase Activity Sensitizes Retinoblastoma-Deficient Cells to DNA Damage–Induced Necrosis

Background: As bulk and single-cell RNA (scRNA) sequencing studies and data continue to accrue to publicly accessible databases, bioinformatic tools continue being developed to analyze these datasets. The resulting pipelines often focus on either bulk or scRNA sequencing but rarely both. scRNA sequencing allows for in-depth characterization of transcription level events in distinct cell populations while bulk data offers a more global view, and for now, is more widely available in databases. Alternative splicing events in particular have been underexplored in a single-cell genomic architecture. The purpose of this study was to develop a bioinformatic pipeline to employ both bulk and single-cell mammary datasets to identify and validate alternative splicing events.Methods: Bulk fastq’s were utilized to identify AS events in luminal progenitor (LP) and mouse mammary stem cell (MSC) lineages in ovariectomized FVB mice that had been randomized into three treatment groups (SHAM [C], estradiol + progesterone [EP], EP + the selective progesterone receptor inhibitor telapristone acetate, TPA [EPT]). scRNA sequencing data from (Bach et al., 2017) was downloaded from the Gene Expression Omnibus (GEO) and utilized for validation of the AS events identified in the bulk data. To separate the data by progesterone level, the scRNA data was parsed into nulliparous, gestational, lactational, and post-involution groups. Further, to determine cell lineage level effects, the data was parsed into luminal and basal compartments using krt18 and krt5 expression. To accomplish the overall comparison between bulk and scRNA sequencing data, we developed a pipeline that would process and parse the scRNA data, identify the alternative splicing in bulk sequencing using rMATS and in the scRNA data using Outrigger (part of the Expedition suite). The resulting data from rMATS [bulk] LP/MSC: CvEP, CvEPT, & EPvEPT were filtered for events meeting statistical significance (p-value < 0.05, FDR < 0.01). After filtering, the events from each treatment comparison were then filtered so only events unique to LP or MSC CvEP, respectively, remain. The remaining significant and unique AS events are then compared to the Krt18-high and Krt5-high Outrigger results from each development stage based on genomic event coordinates (with a buffer +/- 20 base pairs).Results: 12 alternative splicing (AS) events were identified to be shared between the bulk and scRNA sequencing data in the context of increased progesterone exposure (EP and gestational, respectively) as well as cell lineage (LP and luminal respectively). Among the genes identified as having been alternatively spiced with an exact nucleotide match for the splice were Eif4a2, Pik3c2a, Brd4, Cdh1, and Enah. Conclusions: Alternative splicing events can be identified and validated between genomic sequencing methods, specifically bulk and single-cell RNA seq. We hypothesize that as scRNA-seq becomes more developed and quantification errors caused by short-read lengths fixed by full length scRNA seq methods currently in development, comparing data between single-cell and bulk may be made easier. However, we have shown that, when orthogonal methods for validation may not be feasible, events as specific as alternative splices can be validated using publicly available data and in-silico methods. Citation Format: Gannon Cottone, Benjamin T Spike, Elnaz Mirzaei Mehrabad, Seema A Khan, Susan Clare. Validating alternative splicing events between bulk and scRNA sequencing data: A bioinformatic approach [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-16.

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Purpose:Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel–Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC.Experimental Design:To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC.Results:HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs.Conclusions:These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel–Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. Experimental Design: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. Results: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. Conclusions: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

Parallels among embryonic development, stem cells, and cancer have long been recognized. We identified, isolated, and characterized stem cells that first become committed to a mammary fate during embryogenesis; we refer to these cells as fetal mammary stem cells (fMaSCs). Lineage tracing, in vitro sphere formation, and in vivo transplantation studies by our group and many others all confirm that cells in the embryo are the bipotent progenitors of the mammary gland. There is debate, however, on whether such bipotent cells persist into the adult, or whether the luminal and basal lineages are maintained by unipotent progenitors. To gain insight into the relationships between fMaSCs and breast cancer, and to investigate their potential persistence in the adult, we have applied bulk and single cell RNA-sequencing (sc-RNA-seq) and single nucleus ATAC-sequencing (snATAC-seq) throughout mammary development. The results to be discussed demonstrate that fMaSC transcriptomes are heterogeneous, but all share co-expression of genes associated with luminal and basal cell fates. This fits a model in which the bipotent state is created by a balance of lineage specifiers. We also find that the fMaSC transcriptome is highly enriched in basal-like human breast cancers and identify potential embryonic pathways that correlate with poor prognosis. We used a variety of computational tools to infer the gene expression programs that ensue when fMaSCs commit to luminal and basal states. The data from scRNA-seq and snATAC-seq demonstrate that the transitions are gradual, not precipitous, and that luminal and basal cells exhibit significant transcriptomic and epigenetic heterogeneity. This challenges the notion that the mammary gland consists of discrete cell types defined by rigid transcriptomic parameters, and reveals a potential for intrinsic phenotypic plasticity of normal mammary cells. Using the combined databases, we identified Sox10 as a significantly differentially expressed cell state regulator. We show that tumors are heterogeneous with regard to Sox10 expression, and that locally invasive cells tend to express high Sox10 levels. Elevated Sox10 correlates with acquisition of a neural-crest like, EMT-related state. Implications for interception of metastasis by targeting neural crest-like cells will be discussed. Finally, we have generated a web resource that is available to the scientific community to enable the transcription and epigenetic characteristics of any gene of interest to be tracked through mammary development (https://wahl-labsalk.shinyapps.io/Mammary_snATAC/). Citation Format: GM Wahl, Z Ma, C Chung, C Dravis, BT Spike, RR Giraddi, O Balcioglu, C Fan, B Hagos, R Heinz, Herrera-Valdez J, X Hou, J Hwang, R Lasken, G Luna, NE Lytle, EM Mehrabad, M Novotny, CM Perou, O Poirion, S Preissl, B Ren, T Reya, CL Trejo, KT Varley. Understanding breast cancer using a developmental perspective [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES10-2.

Summary DACH1 abundance is reduced in human malignancies, including breast cancer. Herein DACH1 was detected among multipotent fetal mammary stem cells in the embryo, among mixed lineage precursors, and in adult basal cells and (ERα+) luminal progenitors. Dach1 gene deletion at 6 weeks in transgenic mice reduced ductal branching, reduced the proportion of mammary basal cells (Lin− CD24med CD29high) and reduced abundance of basal cytokeratin 5, whereas DACH1 overexpression induced ductal branching, increased Gata3 and Notch1, and expanded mammosphere formation in LA-7 breast cells. Mammary gland-transforming growth factor β (TGF-β) activity, known to reduce ductal branching and to reduce the basal cell population, increased upon Dach1 deletion, associated with increased SMAD phosphorylation. Association of the scaffold protein Smad anchor for receptor activation with Smad2/3, which facilitates TGF-β activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-β activity in vivo., Highlights • Dach1 is expressed in mammary gland fetal stem cells and adult luminal cells • Dach1 expands mammary gland basal/myoepithelial cells • Dach1 induces post-natal mammary gland ductal formation • Dach1 retrains TGF-β activity in the mammary gland in vivo, DACH1 abundance, is reduced in human malignancies, including breast cancer. In this article, Dr. Pestell and his colleagues showed that DACH1 is expressed in multipotent mammary gland fetal stem cells and both the adult basal and ERα+ luminal cells, promotes mammary gland stem cell and basal/myoepithelial cell expansion, promotes mammary gland ductal branching, and restrains TGF-β action in the pubertal mammary gland.

Changes in cellular identity (also known as histologic transformation or lineage plasticity) can drive malignant progression and resistance to therapy in many cancers, including lung adenocarcinoma (LUAD). The lineage-specifying transcription factors FoxA1 and FoxA2 (FoxA1/2) control identity in NKX2-1/TTF1-negative LUAD. However, their role in NKX2-1-positive LUAD has not been systematically investigated. We find that Foxa1/2 knockout severely impairs tumorigenesis in KRAS-driven genetically engineered mouse models and human cell lines. Loss of FoxA1/2 leads to the collapse of a dual-identity state, marked by co-expression of pulmonary and gastrointestinal transcriptional programs, which has been implicated in LUAD progression. Mechanistically, FoxA1/2 loss leads to aberrant NKX2-1 activity and genomic localization, which in turn actively inhibits tumorigenesis and drives alternative cellular identity programs that are associated with non-proliferative states. This work demonstrates that FoxA1/2 expression is a lineage-specific vulnerability in NKX2-1-positive LUAD and identifies mechanisms of response and resistance to targeting FoxA1/2 in this disease.

Change in cancer cell identity is well characterized as a mechanism of cancer progression and acquired resistance to targeted therapies. Lung adenocarcinoma (LUAD) exhibits significant heterogeneity in cell identity and differentiation state; these characteristics correlate directly with prognosis, response to available therapies, and acquisition of drug resistance. In previous work, we have shown that FoxA1 and FoxA2 (FoxA1/2) activate a gastric differentiation program in NKX2-1-negative LUAD. Here we investigate the role of FoxA1/2 in NKX2-1-positive LUAD. We find that FoxA1/2 are consistently expressed in NKX2-1-positive human LUAD. Foxa1/2 deletion severely impairs proliferation and significantly prolongs overall survival in a genetically engineered mouse model of KRAS-driven LUAD. FoxA1/2 activate expression of a mixed-lineage transcriptional program characterized by co-expression of pulmonary (Alveolar Type II) and gastrointestinal marker genes. Loss of FoxA1/2 causes a lineage switch, activating gene expression programs associated with Alveolar Type I cells and maturing squamous epithelial cells. Inhibition of NKX2-1 partially rescues the antiproliferative impact of FoxA1/2 loss, showing that NKX2-1 retains some degree of activity in FoxA1/2-null cells, despite its inability to activate canonical target genes. In summary, this study identifies FoxA1/2 expression as a novel vulnerability in NKX2-1 positive LUAD and shows that FoxA1/2 actively regulate cellular identity in this disease.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies., eLife digest When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue. As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell. How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment. Many lung cancers have an identity similar to normal lung cells. However, some turn off a gene that codes for a protein called NKX2-1, which leads to a type of cancer called invasive mucinous adenocarcinoma (or IMA for short). Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach. But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1, and how transitioning to a different cell type impacts their response to treatment. To investigate this, Zewdu et al. studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer. This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors. Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer. These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell. This identity change was found to depend on two signaling pathways which cells use to communicate. This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors. It also shows new relationships between key genes in these cancers and systems for cell communication. These findings could lead to better therapies for lung cancer, particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment.

CD146 (Mcam; Muc18; s-endo; and gicerin) is an integral membrane glycoprotein originally identified in a human malignant melanoma, but mis-expressed in a wider variety of malignancies including breast cancer, where it has been implicated in controlling tumor growth and dissemination. The mechanisms by which CD146 conveys extracellular cues into malignant cellular outputs is not well understood, but its high-level expression in early development and normal mouse mammary stem cells, as well as expression in highly plastic adult pericytes (formerly known as mesenchymal stem cells) suggested to us that it may control cell state plasticity. We tested the role of CD146 in a plastic, stem cell-like mouse mammary tumor cell line derived from the MMTV-PyMT mouse (PY230). We identified domains in the CD146 cytoplasmic tail essential for controlling substrate utilization by upstream kinases in Akt, Mapk, Stat3, and integrin-related signaling pathways that alter cell state plasticity and tumorigenicity. This work suggests that MCAM plays a key role in controlling lineage plasticity in normal and neoplastic mammary cells with relevance for understanding tumor cell plasticity, progression and evasion of targeted cancer therapies. Citation Format: Ozlen Balcioglu, Brooke L. Gates, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1531.

Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Background CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. Methods We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. Results ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. Conclusions Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

Cancer cells often undergo lineage switching during their natural progression and in response to therapy. Lung adenocarcinomas (LUADs) exhibit a variety of differentiation states accompanied by dysregulation of lineage-specific transcription factors such as NKX2-1. Loss of NKX2-1 in human and murine LUAD leads to invasive mucinous adenocarcinoma (IMA), a subtype of lung cancer that exhibits pulmonary to gastric transdifferentiation. Human IMAs harbor a distinct spectrum of mutationally activated driver oncogenes compared to LUAD overall, suggesting that the transdifferentiation induced by NKX2-1 loss plays a context-dependent role in LUAD progression. Using genetically engineered mouse models, we find that NKX2-1 is required for optimal BRAF V600E driven lung tumor initiation but is dispensable for growth of established lung tumors. NKX2-1-deficient, BRAF V600E driven tumors morphologically resemble human IMA, have high levels of ERK phosphorylation and exhibit a distinct response to treatment with combined BRAF/MEK inhibitors. Whereas NKX2-1-positive tumor cells enter quiescence when treated with BRAF/MEK inhibitors, residual NKX2-1-negative cells fail to exit the cell cycle in response to the same therapy. Additionally, BRAF/MEK inhibitors induce canonical WNT signaling in NKX2-1-negative lung tumor cells, which is accompanied by cell identity switching within the gastric lineage. Co-inhibition of MAPK and WNT pathways blocked elements of this lineage switch in vitro and interfered with cell cycle changes imposed by MAPK inhibition in vivo. Our data show that there is a complex and reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of LUAD identity and suggest that lineage switching induced by targeted therapies may confer new therapeutic vulnerabilities.

Small cell lung cancer (SCLC) is a neuroendocrine tumor treated clinically as a single disease with poor outcomes. Distinct SCLC molecular subtypes have been defined based on expression of ASCL1, NEUROD1, POU2F3, or YAP1. Here, we use mouse and human models with a time-series single-cell transcriptome analysis to reveal that MYC drives dynamic evolution of SCLC subtypes. In neuroendocrine cells, MYC activates Notch to dedifferentiate tumor cells, promoting a temporal shift in SCLC from ASCL1+ to NEUROD1+ to YAP1+ states. MYC alternatively promotes POU2F3+ tumors from a distinct cell type. Human SCLC exhibits intratumoral subtype heterogeneity, suggesting that this dynamic evolution occurs in patient tumors. These findings suggest that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype.

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Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor that is treated clinically as a single disease with poor outcomes. However, SCLC is recently recognized to comprise multiple molecular subsets with unique therapeutic vulnerabilities. Four distinct subtypes of SCLC have been defined based on expression of lineage-related transcription factors: ASCL1, NEUROD1, POU2F3 or YAP1. The origins of these subtypes remain unknown. We use mouse and human SCLC models with a time-series analysis of single-cell transcriptome profiling to reveal that the oncogene MYC drives the dynamic evolution of SCLC subtypes by activation of Notch signaling. MYC cooperates with Notch signaling to promote a temporal shift from an ASCL1-to-NEUROD1-to-YAP1-positive state from a neuroendocrine cell of origin, whereas MYC promotes POU2F3+ tumors from a distinct cell type. SCLC molecular subtypes are therefore not distinct, but rather represent dynamic stages of MYC-driven tumor evolution. Treatment-naive human SCLC exhibits intratumoral heterogeneity in SCLC subtypes, suggesting this dynamic evolution occurs in patient tumors. These findings demonstrate that genetics, cell of origin, and tumor cell plasticity determine SCLC subtype. Given the reported unique therapeutic vulnerabilities of each subtype, we postulate that SCLC tumors represent a “moving therapeutic target” that may require more general, combinatorial, or plasticity-directed therapeutic approaches to combat this transcriptional flexibility. We anticipate that molecular subsets of other cancer types may also represent dynamic stages of tumor evolution. Citation Format: Abbie S. Ireland, Alexi M. Micinski, David W. Kastner, Bingqian Guo, Sarah J. Wait, Kyle B. Spainhower, Christopher C. Conley, Opal S. Chen, Matthew R. Guthrie, Danny Soltero, Yi Qiao, Xiaomeng Huang, Szabolcs Tarapcsak, Siddhartha Devarakonda, Milind D. Chalishazar, Jason Gertz, Justin C. Moser, Gabor Marth, Sonam Puri, Benjamin L. Witt, Benjamin T. Spike, Trudy G. Oliver. MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-120.

SummaryTo discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.

SUMMARY The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis., Graphical Abstract, In Brief Single-cell RNA sequencing of developing mouse mammary epithelia reveals the timing of lineage specification. Giraddi et al. find that fetal mammary stem cells co-express factors that define distinct lineages in their progeny and bear functionally relevant metabolic program signatures that change with differentiation and are resurrected in human breast cancers and metastases.

The search for the bipotent mammary stem cells that drive mammary development requires markers to enable their prospective isolation. There is general agreement that bipotent mouse mammary stem cells are abundant in late fetal development, but their existence in the adult is vigorously debated. Among markers useful for mammary stem cell identification, the Wnt co-receptor Lgr5 has been suggested by some to be both “necessary and sufficient” for bipotency and transplantation of adult mammary stem cell activity, though other studies disagree. Importantly, the relevance of Lgr5 to the bipotency of fetal mammary stem cells has not been studied. We show here that expression of a fluorescent protein driven by the endogenous Lgr5 promoter enables significant fetal mammary stem cell enrichment. We used lineage tracing to demonstrate embryonic cells expressing Lgr5 are bipotent, while their adult counterparts are myoepithelial restricted. Importantly, our data conclusively demonstrate that Lgr5 is dispensable for both fetal and adult mammary stem cell activity and for the development of mammary tumors., Stem cells: protein not needed for stem cell activity in mammary gland A protein considered necessary for mammary stem cell activity turns out to be dispensable. What’s more, it’s not required for tumor development either. Geoffrey Wahl, Christy Trejo, and colleagues from the Salk Institute for Biological Studies in La Jolla, CA, USA, investigated whether the expression of a protein called Lgr5 denotes mammary stem cells and whether it’s truly needed for the cells to be capable of giving rise to two cell lineages—a subject of active debate among researchers. Wahl’s team showed that mouse fetal mammary stem cells expressing Lgr5 had this dual-lineage capacity; their counterparts in the adult mouse did not. Yet, even though Lgr5 expression marks a population of fetal mammary stem cells, the protein is not required for stem cell activity or for tumors to form in a mouse model of breast cancer.

Summary Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell-surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers., Graphical Abstract, Highlights • CRIPTO/GRP78 signaling activates PI3K/AKT in fetal mammary epithelial cells ex vivo • Cell-surface GRP78 marks a CRIPTO-responsive adult mammary stem cell population • An antagonist, ALK4L75A-Fc, blocks soluble CRIPTO growth-factor-like activity • CRIPTO promotes and ALK4L75A-Fc inhibits mammary stem cell maintenance ex vivo, Little is known about the niche factors governing mammary stem cell function and if they are conserved throughout mammary gland development and homeostasis. Here, Gray and colleagues identify CRIPTO as a stem cell maintenance factor in fetal and adult mammary stem/progenitor cells and show that its cell-surface receptor, GRP78, is a mammary stem cell marker and determinant of CRIPTO responsiveness.

The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.

Cellular heterogeneity in cancer represents a significant challenge. In order to develop effective and lasting therapies, it is essential to understand the source of this heterogeneity, and its role in tumor progression and therapy resistance. Here, we consider not only genetic and epigenetic mechanisms, but also inflammation and cell state reprogramming in creating tumor heterogeneity. We discuss similarities between normal mammary epithelial developmental states and various breast cancer molecular sub-types, and the cells that are thought to propagate them. We emphasize that while stem cell phenotypes and mesenchymal character have often been conflated, existing data suggest that the combination of intrinsic genetic and epigenetic changes, and microenvironmental influences generate multiple types of tumor propagating cells distinguishable by their positions along a continuum of epithelial to mesenchymal, stem to differentiated and embryonic to mature cell states. Consequently, in addition to the prospect of stem cell-directed tumor therapies, there is a need to understand interrelationships between stem cell, epithelial–mesenchymal, and tumor-associated reprogramming events to develop new therapies that mitigate cell state plasticity and minimize the evolution of tumor heterogeneity.

Breast cancer stem cells (BCSCs) were the first cancer stem cells defined among solid tissue malignancies. In a little over a decade since their discovery, the literature has boomed with additional markers and regulatory mechanisms governing their behavior. However, it has become evident in that time that BCSCs may themselves be heterogeneous and plastic. This calls for a more comprehensive but also higher-resolution investigation of the various cells that comprise a cancer and will potentially require new paradigms to describe their cellular hierarchies and the role of stem cell biology within them.

Instructors in transformed educational environments face an increased demand to anticipate, engage with, and build upon student ideas in the classroom. Consequently, there is a need to augment traditional forms of preparation to accommodate the expanding role of instructors in these environments. We document a model of weekly preparation designed to engage graduate teaching assistants (TAs) and undergraduate learning assistants (LAs) in developing broader teaching skills through the weekly discussion of student difficulties. In this paper, we describe this model and present evidence of success at focusing instructor attention on student difficulties, accomplished within existing institutional structures and without adding significantly to instructor preparation time.

Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and co-express multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal significant links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis and therapy.

Breast cancer comprises a heterogeneous set of diseases distinguishable from one another by pathologic presentation and molecular signatures. However, each breast cancer subtype is also heterogeneous. Some of the heterogeneity may be attributable to genetic instability, but recent data emphasize that developmental plasticity may also contribute. The p53 tumor suppressor could constitute a nodal control point underlying both sources of heterogeneity because it is frequently inactivated during malignant progression and has recently been shown to function as a potent barrier preventing fully differentiated cells from reverting to pluripotent stem cells after expression of appropriate oncogenes. Using archival microarray datasets, we tested the hypothesis that a p53 mutation could allow cells within a tumor to acquire a stem cell-like state by looking for coordinate expression of stem cell identity genes. We show that breast and lung cancers with p53 mutations do exhibit stem cell-like transcriptional patterns. Such tumors were also depleted for differentiation genes regulated by the polycomb repressor complex 2. These data are consistent with a model in which loss of p53 function enables acquisition of stem cell properties, which are positively selected during tumor progression.

Normal production of RBCs requires that the antiapoptotic protein Bcl-xl be induced at end stages of differentiation in response to erythropoietin (Epo) signaling. The critical proapoptotic pathways inhibited by Bcl-xl in erythroblasts are unknown. We used gene targeting in the mouse to evaluate the BH3-only factor Nix, which is transcriptionally up-regulated during Epo-stimulated in vitro erythrocyte differentiation. Nix null mice are viable and fertile. Peripheral blood counts revealed a profound reticulocytosis and thrombocytosis despite normal serum Epo levels and blood oxygen tension. Nix null mice exhibited massive splenomegaly, with splenic and bone marrow erythroblastosis and reduced apoptosis in vivo during erythrocyte maturation. Hematopoietic progenitor populations were unaffected. Cultured Nix null erythroid cells were hypersensitive to Epo and resistant to apoptosis stimulated by cytokine deprivation and calcium ionophore. Transcriptional profiling of Nix null spleens revealed increased expression of cell cycle and erythroid genes, including Bcl-xl, and diminished expression of cell death and B cell-related genes. Thus, cell-autonomous Nix-mediated apoptosis in opposition to the Epo-induced erythroblast survival pathway appears indispensable for regulation of erythrocyte production and maintenance of hematological homeostasis. These results suggest that physiological codependence and coordinated regulation of pro- and antiapoptotic Bcl2 family members may represent a general regulatory paradigm in hematopoiesis.

The Rb tumor suppressor pathway is functionally inactivated in most human cancers. In human blood cancers, loss of Rb expression has been widely reported in both acute myeloblastic and acute lymphoblastic leukemias, cyclin D1 amplification is common in mantle cell lymphoma, and silencing of the p16/INK4a tumor suppressor gene occurs frequently in AML and various types of lymphoma. Silencing of p16/INK4A expression is frequently caused by hyper-methylation of CpG islands in its promoter but may also be achieved through activation of Bmi-1, for example in lymphomas associated with the E2A-Pbx1 translocation. Hematopoietic homeostasis ensures a balance between proliferation and differentiation at different levels within the hematopoietic hierarchy such that hematopoietic stem cell self-renewal, progenitor proliferation and terminal differentiation to functional blood cells are maintained throughout the life of the animal. Leukemic transformation disrupts this balance favoring proliferation over differentiation, but the mechanisms underlying cell cycle regulation and checkpoint control in the hematopoietic system are not understood. Recent data from our laboratory has identified a unique role for the Rb tumor suppressor gene in the response to anemic and oxidative stress that has implications not just for red cell production but for the homeostatic balance in the entire hematopoietic system and for the development of hematopoietic malignancies.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence. There is growing evidence that stromal p53 also exerts anti-tumor activity by paracrine mechanisms, though a role for stromal p53 in PDAC has not yet been described. Here, we demonstrate that activation of stromal p53 exerts anti-tumor effects in PDAC. We show that primary cancer-associated PSCs (caPSCs) isolated from human PDAC express wild-type p53, which can be activated by the Mdm2 antagonist Nutlin-3a. Our work reveals that p53 acts as a major regulator of PSC activation and as a modulator of PDAC fibrosis. In vitro, p53 activation by Nutlin-3a induces profound transcriptional changes, which reprogram activated PSCs to quiescence. Using immunofluorescence and lipidomics, we have also found that p53 activation induces lipid droplet accumulation in both normal and tumor-associated fibroblasts, revealing a previously undescribed role for p53 in lipid storage. In vivo, treatment of tumor-bearing mice with the clinical form of Nutlin-3a induces stromal p53 activation, reverses caPSCs activation, and decreases fibrosis. All together our work uncovers new functions for stromal p53 in PDAC.

Glucose Regulated Protein (GRP) 94 and GRP78 are critical molecular chaperones and regulators of signaling. Conditional knockout mouse models have revealed tissue specific requirements for GRP94 and GRP78, including selection for allele retention in specific cell types. Here we report the consequences of mammary-targeted knockout of these GRPs. Our studies revealed that MMTV-Cre, Grp94(f/f) mammary glands, despite GRP94 deficiency, exhibited normal proliferation and ductal morphogenesis. Interestingly, MMTV-Cre, Grp78(f/f) mammary glands displayed only slightly reduced GRP78 protein levels, associating with the retention of the non-recombined Grp78 floxed alleles in isolated mammary epithelial cells and displayed phenotypes comparable to wild-type glands. In contrast, transduction of isolated Grp78(f/f) mammary epithelial stem/progenitor cells with adenovirus expressing GFP and Cre-recombinase was successful in GRP78 ablation, and the GFP sorted cells failed to give rise to repopulated mammary glands in de-epithelialized recipient mice. These studies imply GRP78, but not GRP94, is required for mammary gland development.

Kawasaki Disease (KD) is a potentially fatal acute vasculitis of childhood. Although KD is the leading cause of acquired heart disease in children in developed nations, its pathogenesis remains unknown. We previously reported the novel observation that IgA plasma cells infiltrate the vascular wall in acute KD. We have now examined the clonality of this IgA response in vascular tissue from three fatal cases of KD to determine whether it is oligoclonal, suggesting an Ag-driven process, or polyclonal, suggesting nonspecific B cell activation or a response to a superantigen. We first sequenced VDJ junctions of 44 α genes isolated from a primary, unamplified KD vascular cDNA library. Five sets of clonally related α sequences were identified, comprising 34% (15 of 44) of the isolated α sequences. Furthermore, point mutations consistent with somatic mutation were detected in the related sequences. Next, using formalin-fixed coronary arteries from two additional fatal KD cases, we sequenced VDJ junctions of α genes isolated by RT-PCR, and a restricted pattern of CDR3 usage was observed in both. We conclude that the vascular IgA response in acute KD is oligoclonal. The identification of an oligoclonal IgA response in KD strongly suggests that the immune response to this important childhood illness is Ag-driven.

Teaching Assistants (TAs) play an important role in supporting research-based instructional environments, yet the connection between TAs’ pedagogical beliefs and instructional practices is not well understood. We build upon a broad foundation of research on the nature of pedagogical content knowledge to develop an analytic framework for characterizing how physics TAs both describe and enact their roles as teachers. In a previous work [1], we started developing a framework for TA pedagogical beliefs and highlighted examples of emergent differences between TAs along one particular dimension, agency. In this paper, we extend this framework to include TAs’ instructional goals, and their methods and purpose of assessment. Using examples drawn from several semesters of interviews and classroom videotape, we apply this framework and describe instances of both strong and weak coordination between TA beliefs and practices.