Kevin Roarty

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo.

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.

Wnt and mammary stem cells: hormones cannot fly wingless

The mammary stem cell and its local microenvironment are central for the maintenance of proper tissue homeostasis during normal development. Defining the hierarchical organization of the epithelial subtypes in the mammary gland and the molecular pathways guiding their development has begun to provide a framework for understanding how cancer stem cells sustain the progression and heterogeneity of breast cancers. The Wnt pathway plays a fundamental role in multiple adult stem cells, as well as in orchestrating proper mammary gland development and maintenance. These processes are intricately guided by the influence of systemic hormones and local factors. Alterations in Wnt signaling can skew the homeostatic balance of the mammary epithelium to drive malignant progression; however, complexities of Wnt pathway components present a challenge in understanding their physiological function.

Fibroblast Growth Factor Receptor Signaling Is Essential for Normal Mammary Gland Development and Stem Cell Function

Fibroblast growth factor (FGF) signaling plays an important role in embryonic stem cells and adult tissue homeostasis, but the function of FGFs in mammary gland stem cells is less well defined. Both FGFR1 and FGFR2 are expressed in basal and luminal mammary epithelial cells (MECs), suggesting that together they might play a role in mammary gland development and stem cell dynamics. Previous studies have demonstrated that the deletion of FGFR2 resulted only in transient developmental defects in branching morphogenesis. Using a conditional deletion strategy, we investigated the consequences of FGFR1 deletion alone and then the simultaneous deletion of both FGFR1 and FGFR2 in the mammary epithelium. FGFR1 deletion using a keratin 14 promoter‐driven Cre‐recombinase resulted in an early, yet transient delay in development. However, no reduction in functional outgrowth potential was observed following limiting dilution transplantation analysis. In contrast, a significant reduction in outgrowth potential was observed upon the deletion of both FGFR1 and FGFR2 in MECs using adenovirus‐Cre. Additionally, using a fluorescent reporter mouse model to monitor Cre‐mediated recombination, we observed a competitive disadvantage following transplantation of both FGFR1/R2‐null MECs, most prominently in the basal epithelial cells. This correlated with the complete loss of the mammary stem cell repopulating population in the FGFR1/R2‐attenuated epithelium. FGFR1/R2‐null MECs were partially rescued in chimeric outgrowths containing wild‐type MECs, suggesting the potential importance of paracrine mechanisms involved in the maintenance of the basal epithelial stem cells. These studies document the requirement for functional FGFR signaling in mammary stem cells during development. STEM Cells2013;31:178–189

STAT3 Signaling Is Activated Preferentially in Tumor-Initiating Cells in Claudin-Low Models of Human Breast Cancer

In breast cancer, a subset of tumor‐initiating cells (TIC) or “cancer stem cells” are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44high/CD24low/neg, ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3‐mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin‐low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP+) is associated with a subpopulation of cancer cells enriched for mammosphere‐forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem‐cell‐directed therapy in some breast cancer subtypes. Stem Cells 2014;32:2571–2582

Pregnancy-induced noncoding RNA (PINC) associates with polycomb repressive complex 2 and regulates mammary epithelial differentiation.

Pregnancy-induced noncoding RNA (PINC) and retinoblastoma-associated protein 46 (RbAp46) are upregulated in alveolar cells of the mammary gland during pregnancy and persist in alveolar cells that remain in the regressed lobules following involution. The cells that survive involution are thought to function as alveolar progenitor cells that rapidly differentiate into milk-producing cells in subsequent pregnancies, but it is unknown whether PINC and RbAp46 are involved in maintaining this progenitor population. Here, we show that, in the post-pubertal mouse mammary gland, mPINC is enriched in luminal and alveolar progenitors. mPINC levels increase throughout pregnancy and then decline in early lactation, when alveolar cells undergo terminal differentiation. Accordingly, mPINC expression is significantly decreased when HC11 mammary epithelial cells are induced to differentiate and produce milk proteins. This reduction in mPINC levels may be necessary for lactation, as overexpression of mPINC in HC11 cells blocks lactogenic differentiation, while knockdown of mPINC enhances differentiation. Finally, we demonstrate that mPINC interacts with RbAp46, as well as other members of the polycomb repressive complex 2 (PRC2), and identify potential targets of mPINC that are differentially expressed following modulation of mPINC expression levels. Taken together, our data suggest that mPINC inhibits terminal differentiation of alveolar cells during pregnancy to prevent abundant milk production and secretion until parturition. Additionally, a PRC2 complex that includes mPINC and RbAp46 may confer epigenetic modifications that maintain a population of mammary epithelial cells committed to the alveolar fate in the involuted gland.

The mammary stem cell hierarchy: a looking glass into heterogeneous breast cancer landscapes

The mammary gland is a dynamic organ that undergoes extensive morphogenesis during the different stages of embryonic development, puberty, estrus, pregnancy, lactation and involution. Systemic and local cues underlie this constant tissue remodeling and act by eliciting an intricate pattern of responses in the mammary epithelial and stromal cells. Decades of studies utilizing methods such as transplantation and lineage-tracing have identified a complex hierarchy of mammary stem cells, progenitors and differentiated epithelial cells that fuel mammary epithelial development. Importantly, these studies have extended our understanding of the molecular crosstalk between cell types and the signaling pathways maintaining normal homeostasis that often are deregulated during tumorigenesis. While several questions remain, this research has many implications for breast cancer. Fundamental among these are the identification of the cells of origin for the multiple subtypes of breast cancer and the understanding of tumor heterogeneity. A deeper understanding of these critical questions will unveil novel breast cancer drug targets and treatment paradigms. In this review, we provide a current overview of normal mammary development and tumorigenesis from a stem cell perspective.

Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium

Intricate cross-talk between classical and alternative Wnt signaling pathways includes an essential role for Ror2 in mammary epithelial development and differentiation.

Paracrine signaling in mammary gland development: what can we learn about intratumoral heterogeneity?

Paracrine signaling mechanisms play a critical role in both normal mammary gland development and breast cancer. Dissection of these mechanisms using genetically engineered mouse models has provided significant insight into our understanding of the mechanisms that guide intratumoral heterogeneity. In the following perspective, we briefly review some of the emerging concepts in this field and emphasize why elucidation of these pathways will be important for future progress in devising new and improved combinatorial therapeutic approaches for breast and other solid cancers.

Ror2-mediated alternative Wnt signaling regulates cell fate and adhesion during mammary tumor progression

Cellular heterogeneity is a common feature in breast cancer, yet an understanding of the coexistence and regulation of various tumor cell subpopulations remains a significant challenge in cancer biology. In the current study, we approached tumor cell heterogeneity from the perspective of Wnt pathway biology to address how different modes of Wnt signaling shape the behaviors of diverse cell populations within a heterogeneous tumor landscape. Using a syngeneic TP53-null mouse model of breast cancer, we identified distinctions in the topology of canonical Wnt β-catenin-dependent signaling activity and non-canonical β-catenin-independent Ror2-mediated Wnt signaling across subtypes and within tumor cell subpopulations in vivo. We further discovered an antagonistic role for Ror2 in regulating canonical Wnt/β-catenin activity in vivo, where lentiviral shRNA depletion of Ror2 expression augmented canonical Wnt/β-catenin signaling activity across multiple basal-like models. Depletion of Ror2 expression yielded distinct phenotypic outcomes and divergent alterations in gene expression programs among different tumors, despite all sharing basal-like features. Notably, we uncovered cell state plasticity and adhesion dynamics regulated by Ror2, which influenced Ras Homology Family Member A (RhoA) and Rho-Associated Coiled-Coil Kinase 1 (ROCK1) activity downstream of Dishevelled-2 (Dvl2). Collectively, these studies illustrate the integration and collaboration of Wnt pathways in basal-like breast cancer, where Ror2 provides a spatiotemporal function to regulate the balance of Wnt signaling and cellular heterogeneity during tumor progression.

Abstract 3306: Wnt pathway dynamics in breast cancer progression

Background: The canonical Wnt/β-catenin signaling pathway directs self-renewal cues for stem cells in multiple tissues. Through alternative receptors, noncanonical Wnt/β-catenin-independent pathways exist, mediating cellular processes that include planar cell polarity, convergent extension, actin/cytoskeletal rearrangements, and in some contexts, antagonism of canonical Wnt signaling. How different Wnt pathways cooperate within heterogeneous cellular landscapes in breast cancers remains an outstanding question. Within the current study, we investigated the role of the alternative Wnt receptor, Ror2, in mediating Wnt/β-catenin-independent functions during tumor progression. Experimental design and methods: A TP53 null transplantable GEM model was used in the current study, which exhibits subtype-specific molecular signatures characteristic of human breast cancers. A lentiviral-based shRNA strategy was developed for gene silencing of noncanonical Wnt pathway components in vivo, with emphasis on the alternative Wnt receptor, Ror2. This strategy was combined with pathway-specific Wnt reporters to survey the interplay of canonical and noncanonical Wnt pathways in vivo. Results: TP53 null mammary tumors differentially expressed the noncanonical Wnt receptor Ror2, with the highest levels of expression evident in basal-like tumors relative to luminal and claudin-low subtypes analyzed. Basal-like tumors also displayed Wnt/β-catenin active populations, which were inversely correlated with Ror2 expression. Lentiviral shRNA silencing of Ror2 within 3 independent basal-like models (T1, T2, 2225L) resulted in elevated Wnt/β-catenin activity along with various phenotypic outcomes, including squamous differentiation. Ror2, therefore, functions to restrain Wnt/β-catenin-dependent signaling in vivo. Despite the similarity in Wnt/β-catenin-dependent signaling outcome upon Ror2 depletion, gene expression profiling of Ror2-intact (shLUC) vs. Ror2-depleted (shRor2) tumors unveiled diverse gene expression changes within basal-like tumor models. Intriguingly, Ror2 silencing within 2225L basal-like tumors resulted in a change in the composition of tumor cell subpopulations. Additionally, gene expression analysis of shLUC vs. shRor2 tumors revealed that genes upregulated in shRor2 tumors were associated with claudin-low features, while genes downregulated were associated with basal-like features, reflecting changes in the cellular landscape upon Ror2 loss. Gene ontology analysis also revealed alterations in actin/cytoskeletal- and cell adhesion-associated genes upon Ror2 depletion. Conclusion: The current study highlights the differential effects of Ror2 loss within TP53 null basal-like models. In particular, Ror2 can regulate the plasticity between basal-like and claudin-low states, impacting cytoskeletal and cell adhesion dynamics. Current efforts are focused on how these changes impact tumor progression and metastasis. Citation Format: Kevin Roarty, Adam D. Pfefferle, Chad J. Creighton, Charles M. Perou, Jeffrey M. Rosen. Wnt pathway dynamics in breast cancer progression. [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 3306.