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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.

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.

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.

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.

In the original version of [this article][1] ([1][2]), the wrong files were supplied for Figs. 5B and C. The figures and related article text have been corrected in the latest online HTML and PDF versions of this article. The authors regret these errors. 1. 1.[↵][3]1. Cowman SJ, 2. Fuja

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.

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.

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.

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.

The ability of cancer cells to adapt to various extrinsic challenges such as nutrient deprivation, hypoxia and chemotherapy may underlie their tumorigenicity as well as their ability to disseminate to distant organs. Therefore, identifying molecular mechanisms that govern such cellular plasticity may be critical to the development of anti-cancer therapies that target situationally defined tumor stem cells in metastasis, resistance, dormancy and recurrence. We considered the GPI-anchored/secreted Cripto protein to be an excellent target in this regard, as it regulates cellular plasticity during normal development including promotion of EMT, the embryonic stem cell state, and reacquisition of the stem cell state in cellular reprogramming. Cripto also regulates tissue specific stem cells including those of the mammary gland as we recently demonstrated. Due to the rarity of tissue stem cells under homeostasis, Cripto protein is generally low or undetectable in normal adult tissues but it is highly expressed in a wide variety of human tumors including breast cancers where its levels correlate with tumor aggressiveness and poor patient outcomes. Furthermore, inhibition of Cripto expression at the transcript level was recently reported to block metastasis in a mouse mammary tumor model. We have used xenograft assays to demonstrate that the requirement for Cripto function during metastasis, and to a lesser extent in primary tumor growth is conserved in human triple negative breast cancer cells. Critically, we demonstrate this through the use of a biologic/recombinant therapeutic protein specifically engineered to target Cripto. This recombinant protein, ALK4L75A-Fc, is comprised of an Fc-domain fused to a mutant extracellular domain of the natural Cripto binding partner and activin/Nodal type I signaling receptor, ALK4. The relatively modest effect of Cripto blockade on bulk primary tumors relative to metastasis and our inability to identify significant inhibitory effects of ALK4L75A-Fc on these cells in standard in vitro assays suggests that Cripto may only be critical for a subset of cancer cells and/or in specific microenvironmental contexts in the course of tumor progression. Consistent with this, we previously reported that Cripto signaling depends on its cell surface interaction with the stress inducible HSP70 family member and ER chaperone, GRP78 (glucose regulated protein, 78 kDa). Stresses including nutrient deprivation and chemotherapy strongly induce GRP78 expression and increase its levels at the cell surface and we find that nutrient deprivation in vitro elicits sensitivity of the MDA-MB-231 breast cancer cell line to inhibition of migration by ALK4L75A-Fc. Together, these results suggest that Cripto/GRP78 signaling promotes cancer cell plasticity and adaptation to microenvironmental challenge and that ALK4L75A-Fc may have therapeutic potential as an inhibitor of tumor cell plasticity. Citation Format: Peter C. Gray, Evan Booker, Berhane Hagos, Masami Furphy, Benjamin T. Spike. Targeting Cripto in breast cancer plasticity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3304.

The retinoblastoma (Rb) tumor suppressor is a key regulator of cell cycle checkpoints but also protects against cell death induced by stresses such as DNA damage and death receptor ligation. We report here that cell death of Rb-deficient cells exposed to key genotoxic agents was associated with increased expression of S phase–specific E2F target genes and cell death consistently occurred in the S phase of the cell cycle. Cell cycle arrest induced by serum starvation prevented S phase entry, attenuated DNA damage, and promoted survival, suggesting that Rb-null cells die due to a failure to prevent S phase entry. DNA damage–induced death of Rb-null cells was associated with nucleotide depletion, higher activity of poly-ADP-ribose-polymerase (Parp), and cell death that was primarily necrotic. Knockdown of Parp-1 or chemical inhibition of Parp activity prevented nucleotide depletion and restored the viability of Rb-deficient cells to wild-type levels. Furthermore, chemical inhibition of Parp activity in vivo attenuated the cytotoxic effects of cisplatin against Rb-deficient tumors, arguing that Parp inhibitors should not be used therapeutically in combination with genotoxic drugs against tumors that are inactivated for the Rb tumor suppressor.(Mol Cancer Res 2009;7(7):1099–109)

Microenvironmental factors are critical regulators of stem cell and tumor cell behavior. However, few stem cell factors have been identified and targeted in breast and other cancers. Using a highly enriched stem cell pool isolated from fetal mammary rudiments and single cell analyses, we have identified the onco-fetal protein Cripto as a potent soluble factor that regulates the mammary stem cell state. Cripto is a GPI-anchored/secreted signaling protein and a known regulator of PI3K/AKT and TGF-beta pathways. Here, we develop a novel Cripto antagonist, ALK4L75A-Fc, which selectively blocks the growth factor-like effects of soluble Cripto. We show that this antagonist promotes the differentiation of mammary stem cells cultured ex vivo while Cripto treatment maintains the stem cell phenotype and yields colonies with enhanced mammary gland reconstitution capacity. We previously discovered that Cripto signaling requires binding to cell surface Glucose Response Protein 78kDa (GRP78). GRP78 is an HSP70 family member that is induced by stresses including hypoxia and glucose deprivation and it is highly expressed in tumors where these conditions prevail. We show here that cell surface GRP78 marks fetal mouse mammary stem cells and a population of bipotent adult mammary epithelial cells that are selectively responsive to soluble Cripto in vitro. GRP78high mammary epithelial cells also harbor substantially higher stem cell activity upon transplantation than GRP78low cells. Consistently, we further show that deletion of GRP78 from adult mammary epithelial cells ex vivo blocks mammary gland reconstitution. Finally, we find that Cripto antagonism with ALK4L75A-Fc inhibits the proliferation of triple negative breast cancer cell lines in vitro and that this antagonist can also inhibit tumor growth in vivo. Since Cripto and GRP78 are both induced by hypoxia and other stressful conditions found in tumors, our data raise the possibility that Cripto/GRP78 signaling may exacerbate breast and other cancers by increasing the number of stem cell-like tumor cells in tumor microenvironments where these conditions predominate. Citation Format: Benjamin T. Spike, Jonathan A. Kelber, Evan Booker, Madhuri Kalathur, Rose Rodewald, Julia Lipianskaya, Justin La, Marielle He, Tracy Wright, Richard Klemke, Geoffrey Wahl, Peter C. Gray. Cripto/GRP78 signaling promotes the stem cell phenotype in normal and neoplastic mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2014-3035

Molecular pathways that guide cell proliferation and programming events during embryonic development to generate specific organs may be aberrantly re-activated in the cancer state of those organs to drive tumor progression. Recently, our laboratory has successfully demonstrated the applicability of this tenet to studies of breast cancer. We have developed an enrichment protocol for fetal mammary stem cells (fMaSCs) from embryonic mammary rudiments, and verified that particular subtypes of breast cancers, including Basal-like triple-negative breast cancers for which targeted therapies do not exist, show strong correlation with expression signatures found in fMaSCs. This suggests that elucidation of the biology behind fMaSC function will yield important insight into better treatment of these aggressive breast cancers. To identify mechanisms that govern fMaSC function, we have performed transcriptome analyses on cell populations enriched for fMaSCs and their surrounding stroma. From this data, we have identified candidate signaling and transcription networks that we are currently validating. Here we describe ongoing work with one candidate to emerge from our analyses, Sox10. Sox10 is specifically expressed in the enriched fMaSC population, and is of particular interest given the well-documented roles for other Sox family transcription factors in: 1) regulating cell fate decisions in embryonic and adult tissue, and 2) mediating the progression of some types of cancer. We show that Sox10 expression specifically correlates with MaSC function in fetal and adult mammary. We further show that Sox10 expression can be utilized to identify the cells maintaining MaSC stem cell properties in culture. Importantly, incorporation of Sox10 into our existing fMaSC purification protocol results in a twofold improvement in fMaSC enrichment. Our discoveries with Sox10 thus represent a molecular focal point around which to build signaling and transcriptional networks underlying fMaSC state and function. They further demonstrate the promise of our approach in identifying novel targets for the development of new therapies for poorly treated cancers. Citation Format: Christopher Dravis, Claire Johns, Benjamin T. Spike, Michelle Southard Smith, Geoffrey M. Wahl. Sox10 expression labels in vitro mammary stem cell activity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B136.

Somatic stem cells govern the development, maintenance and regeneration of tissues, and their dysregulation is associated with diverse pathologies including cancer. Given the significance of these cells both biologically and therapeutically, it is critical to elucidate the signaling mechanisms that dictate their behavior. Our recent work on the fetal mammary gland has dissected complementary roles for the stem cell compartment and stromal cues in fetal mammary stem cell function. We also identified significant similarities between the gene expression profiles of some of the most aggressive and untreatable forms of human breast cancer and those of the mouse fetal stem cell enriched mammary epithelial and stromal fractions. These similarities suggest that stem cell regulatory mechanisms operative during organogenesis, including those specified by the stromal niche, may serve as novel therapeutic targets in these cancers. We also discovered significant gene expression heterogeneity within the fetal stem cell compartment when analyzed at single cell resolution. While such heterogeneity likely reflects the natural diversity of stem and non-stem cell states comprising the system, it confounds the interpretation of expression profiles from mixed populations and the delineation of stem cell specific and niche specific molecular components. Our current objective is to utilize genome wide single cell RNA-Sequencing to hasten the identification of functionally relevant, stem cell specific regulators and markers in the context of this multi-cellular/multi-signal system. Among other candidates, we have identified the Cripto/GRP78 signaling axis as a critical regulator of mammary stem cell activity, and have developed novel molecular genetic tools to temporally control the activity of candidate stem cell regulators in vivo in normal and neoplastic contexts. Integration of these tools promises to deliver a new molecular understanding of stem cell biology in the mammary gland and other systems and of stem cell-like cancer cells. Citation Format: Benjamin T. Spike, Danielle D. Engle, Jennifer C. Lin, Jonathan A. Kelber, Justin La, Samantha K. Cheung, Evan Booker, Rose Rodewald, Christopher Dravis, Peter C. Gray, Geoffrey M. Wahl. Deconstructing stemness from the fetal mammary perspective. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A136.

Mammary stem cells (MaSCs) self-renew and generate the different epithelial cell types of the breast. Impressively, a single MaSC can regenerate an entire functional mouse mammary gland when transplanted into a cleared fat pad. Intriguingly, stem-like cells have been identified in some types of breast cancer using bioinformatic strategies. These observations raise a number of important questions: Do some breast cancers arise by mutations occurring in mammary stem cells, as such cells have a high capacity for self-renewal, proliferation and differentiation? Or, do breast cancers arise by de-differentiation of cells to a stem-like state, as such cells may be particularly able to generate the heterogeneity that fuels tumor progression? If stem cells contribute to cancer, what is the nature of such cells, and can we use this information to devise better treatment strategies or prognostic molecular signatures? Finally, cancer cells that seem particularly competent at generating tumors upon xenografting into nude mice have also been suggested to exhibit stem cell characteristics such as multi-lineage differentiation and self-renewal. Are these “cancer stem cells” mutant stem cells, or a different type of cell that is particularly tumorigenic in the xenograft assay? Here, we integrate approaches involving developmental, stem cell, and cancer biology to gain insight into these challenging questions. We took a new direction to isolate and characterize MaSCs during fetal mammogenesis when undifferentiated cells are expected to be enriched. We microdissected and dissociated fetal mammary cells across different stages of development and conducted limiting dilution, orthotopic mammary transplantation analyses to determine fetal MaSC (fMaSC) frequency. We found that the fMaSCs are not measurable until mid-gestation, when an elongating duct that invades through the adjacent mesenchyme is formed. We used flow sorting to significantly enrich fMaSCs, and then used microarray and single cell RNA-seq to begin to interrogate the molecular pathways that drive their proliferation. We find that the pathways they express, and that are expressed by the fetal stroma that surround them, are enriched in several very aggressive types of human breast cancer, as well as in several genetically engineered mouse models. These data and powerful experimental strategies are opening new ways of understanding cancer from a developmental perspective, and for using this knowledge to develop new diagnostic and treatment strategies. Citation Format: Geoffrey Myles Wahl, Christopher Dravis, Claire Johns, Rose Rodewald, Benjamin T. Spike, Christy Trejo. Dissecting cancer through the lens of development. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA11.

We recently determined that a fraction of cells in a highly enriched fetal mammary stem cell pool expresses the oncofetal protein Cripto and its receptor/co-factor GRP78 on their surfaces. Cripto is a GPI-anchored protein important for embryonic stem cell self-renewal and developmental plasticity. It is also selectively overexpressed in multiple human tumors including ∼80% of human breast tumors. Cripto overexpression increases cellular proliferation, invasion and migration in vitro and promotes mammary tumor growth in mice. Cripto functions as an obligatory co-receptor for Smad2/3 activation by the TGF-β superfamily member Nodal but attenuates activin-A and TGF-β1 signaling and the cytostatic effects of these ligands on human mammary epithelial cells. Cripto can be secreted from cells and soluble forms activate Src, ras/raf/MAPK and PI3K/Akt pathways. We recently identified the HSP70 family member GRP78 as a cell surface receptor/co-factor that is required for Cripto signaling via TGF-β and Src/MAPK/PI3K pathways. Here, we developed a soluble and highly specific Cripto antagonist based on the extracellular domain of the Activin/Nodal Type I serine/threonine kinase receptor ALK4 (sALK4-ECD). We tested the ability of this antagonist to block Cripto signaling via TGF-β and Src/MAPK/PI3K pathways in oncogenic and stem cell phenotypes in mammary epithelial cells and breast cancer cell lines. The sALK4-ECD protein dose-dependently and specifically inhibited 125I-Cripto binding to 293T cells transfected with full length ALK4. In addition, sALK4-L75A-Fc blocked 125I-Cripto binding to native human mammary epithelial MCF10A cells. sALK4-ECD inhibited Cripto-dependent Nodal signaling in 293T cells and inhibited Nodal-induced Smad2 phosphorylation in native NCCIT cells. sALK4-ECD also blocked Cripto-induced PI3K activation, cell proliferation and migration in MCF10A cells. Cripto and Nodal signaling had profound effects on MCF10A growth in suspension and mammosphere reseeding capacity that were also inhibited by sALK4-ECD. We evaluated Nodal/Cripto signaling in cancer cell lines and primary fetal mammary stem cells and tested the efficacy of sALK-ECD mediated suppression of Nodal and Cripto effects in vitro and in vivo. These studies were designed to test if Cripto represents a functional cell surface marker of normal stem cells and/or the cells that initiate or perpetuate breast cancers. Our results point to distinct roles for Cripto and Nodal/Cripto signaling in promoting the oncogenic phenotype via activation of Smad2/3 and PI3K pathways, and support a role for Cripto antagonism such as that afforded by sALK4-ECD as a potential therapeutic strategy for the treatment of human disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3493. doi:1538-7445.AM2012-3493

Parallels between embryonic development, stem cells, and cancer have long been recognized. The proliferative and invasive characteristics of the most lethal forms of breast cancer and their lack of differentiated characteristics resemble aspects of embryonic mammogenesis, raising the possibility that they share molecular mechanisms. We have identified, isolated and characterized a fetal mammary population with concentrated mammary stem cell activity and shown that it exhibits gene expression similarities to certain human breast cancer molecular subtypes. Limiting dilution transplantation analyses demonstrate that a robust increase in fetal mammary stem cell (fMaSC) activity is evident after embryonic day 16 (E16), coincident with invasion of the rudiment through the mesenchyme and into the fat pad precursor. Markers used to enrich adult MaSCs enabled isolation of a population at E18.5 that is highly enriched for fMaSCs and capable of serial transplantation in vivo and clonal, multilineage sphere formation in vitro. Genome-wide expression profiling and single cell analyses revealed candidate pathways in both the epithelial and stromal compartments with relevance to fMaSC activity and cancer. Notably, ErbB and FGF signaling were critical and synergistic for the in vitro growth of spheres derived from the fMaSC-enriched population, and fMaSCs expressed elevated ErbB2, 3, and 4 relative to adult MaSC enriched populations. We explored molecular links between fetal mammary development and cancer further by analyzing the expression of fetal gene signatures in archived breast cancer array data and in breast cancer cell lines. We identified gene expression modules correlating with certain cell lines, intrinsic breast cancer subtypes and clinical outcome. These studies advance the attractive hypothesis that regulatory pathways governing fMaSC function and embryonic mammogenesis that are deregulated in certain types of human cancer can be targeted in the development of new therapies and prognostic metrics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1015. doi:1538-7445.AM2012-1015

Mammary stem cells (MaSCs) play a key role in the development and maintenance of the breast. MaSCs self-renew and generate the different cell types of the breast such that a single MaSC can regenerate an entire functional mouse mammary gland. Similarly, tumors are heterogeneous, with some cells exhibiting potent tumor forming ability in transplantation studies. These cells exhibit the stem cell properties of self-renewal and multi-lineage differentiation in that they can serially regenerate the cellular complexity of the original tumor. These cells, referred to as cancer stem cells (CSCs), have in some cases been shown to be more resistant to cancer therapies. Therefore, if even a single CSC survives chemotherapy, it could result in cancer relapse. Consequently, an important goal is to be able to prospectively identify CSCs and elucidate critical growth control pathways to enable development of effective targeted therapies. Unfortunately, pure MaSCs or breast CSCs have not been isolated, precluding the generation of reagents to detect or eliminate them. My research takes a new direction to identify, isolate, and characterize MaSCs using properties central to mammary gland development and stem cell function. We isolated MaSCs during development and found that they are naturally enriched late in embryogenesis. We sorted the mammary cells at E18.5 using cell surface proteins we previously found to be expressed in the rudiment at E18.5 and were able to obtain MaSCs purified to a significantly higher degree than reported in the literature. These fetal MaSCs express several genes implicated in cancer, including erbB2, which is found in a subset of dangerous human cancers. In addition, like human cancers that over-express erbB2, fetal MaSC growth is inhibited by the erbB2 antagonist Lapatinib. The MaSC population isolated during fetal development is enriched for epithelial cells expressing both myoepithelial and luminal keratin markers as well as epithelial cells expressing neither of these markers. It is possible that either of these sub-populations contains the stem cell activity. We have focused on using high throughput single cell QPCR analysis to cluster the fetal MaSCs by their keratin marker expression pattern and identify cell surface markers that can be used to isolate viable cells for stem cell activity assays. Altogether, these experiments enabled isolation of highly enriched MaSC populations for elucidation of key growth pathways and set the stage for future in vivo analyses. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-02.

Cripto is a small GPI-anchored glycoprotein that plays key roles during normal development and in tumorigenesis. Cripto is expressed at aberrantly high levels in human tumors and blocking its oncogenic effects on activin/Nodal/TGF-beta, Erk/MAPK and PI3K/Akt pathways represents a promising therapeutic strategy. Cripto functions as a Nodal co-receptor and binds directly to the extracellular domain (ECD) of the activin/Nodal type I receptor ALK4. We have shown that a kinase-deleted ALK4 mutant deficient in activin binding (ALK4-L75A) retains Cripto binding and can block Cripto effects on activin and Nodal signaling. We hypothesized that soluble ALK4-L75A ECD proteins would similarly bind Cripto and disrupt oncogenic Cripto function. To test this, we generated two soluble constructs incorporating the ALK4-L75A ECD, one with a C-terminal His tag (sALK4-L75A-His) and the other consisting of an ALK4-L75A ECD-Fc fusion with an N-terminal FLAG tag (sALK4-L75A-Fc). We expressed the sALK4-L75A-His and sALK4-L75A-Fc proteins in 293T cells and then purified them from conditioned media using Ni-NTA agarose and Protein-A agarose, respectively. The sALK4-L75A-Fc protein dose-dependently inhibited 125I-Cripto binding to 293T cells transfected with full length ALK4. In addition, sALK4-L75A-Fc blocked 125I-Cripto binding to untransfected human mammary epithelial MCF10A cells. In accordance with these binding data, sALK4-L75A-His inhibited Cripto-dependent Nodal signaling in 293T cells and sALK4-L75A-Fc inhibited Nodal-induced Smad2 phosphorylation in human NCCIT cells. sALK4-L75A-His also blocked Cripto-induced PI3K signaling while sALK4-L75A-Fc inhibited Cripto-induced MCF10A cell proliferation. Importantly, we have shown that sALK4-L75A-Fc can also inhibit the growth of tumor xenografts derived from NCCIT cells at doses where no toxicity was observed. Thus, we provide the first evidence that soluble forms of ALK4 inhibit oncogenic Cripto signaling. The efficacy of a soluble ALK4 receptor as an antitumor agent points to the utility of Cripto antagonists in the treatment of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4006.

Mammary gland morphology and physiology are supported by an underlying cellular differentiation hierarchy. Molecular features associated with particular cell types along this hierarchy may contribute to the biological and clinical heterogeneity observed in human breast carcinomas. Investigating the normal cellular developmental phenotypes in breast tumors may provide new prognostic paradigms, identify new targetable pathways, and explain breast cancer subtype etiology. We used transcriptomic profiles coming from fluorescence-activated cell sorted (FACS) normal mammary epithelial cell types from several independent human and murine studies. Using a meta-analysis approach, we derived consensus gene signatures for both species and used these to relate tumors to normal mammary epithelial cell phenotypes. We then compiled a dataset of breast cancer patients treated with neoadjuvant anthracycline and taxane chemotherapy regimens to determine if normal cellular traits predict the likelihood of a pathological complete response (pCR) in a multivariate logistic regression analysis with clinical markers and genomic features such as cell proliferation. Most human and murine tumor subtypes shared some, but not all, features with a specific FACS-purified normal cell type; thus for most tumors a potential distinct cell type of ‘origin’ could be assigned. We found that both human luminal progenitor and mouse fetal mammary stem cell features predicted pCR sensitivity across all breast cancer patients even after controlling for intrinsic subtype, proliferation, and clinical variables. This work identifies new clinically relevant gene signatures and highlights the value of a developmental biology perspective for uncovering relationships between tumor subtypes and their potential normal cellular counterparts. Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3262-6) contains supplementary material, which is available to authorized users.