Malgorzata Klauzinska

The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition

Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-β (TGF-β) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/β-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.

Developmental signaling pathways regulating mammary stem cells and contributing to the etiology of triple-negative breast cancer

Cancer has been considered as temporal and spatial aberrations of normal development in tissues. Similarities between mammary embryonic development and cell transformation suggest that the underlying processes required for mammary gland development are also those perturbed during various stages of mammary tumorigenesis and breast cancer (BC) development. The master regulators of embryonic development Cripto-1, Notch/CSL, and Wnt/β-catenin play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells (fMaSC) and in the adult organism particularly within the adult mammary stem cells (aMaSC), which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during tumorigenesis to support the tumor-initiating cell (TIC)/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of BC, in particular the most aggressive subtype of BC, triple-negative breast cancer (TNBC). This in depth review will present evidence of the involvement of Cripto-1, Notch/CSL, and Wnt/β-catenin in the normal mammary gland morphogenesis and tumorigenesis, from fMaSC/aMaSC regulation to TIC generation and maintenance in TNBC. Specific therapies for treating TNBC by targeting these embryonic pathways in TICs will be further discussed, providing new opportunities to destroy not only the bulk tumor, but also TICs that initiate and promote the metastatic spread and recurrence of this aggressive subtype of BC.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co‐expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth‐promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co‐transfectants exhibited invasive properties in three‐dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf‐1, a specific antagonist of the Wnt/β‐catenin pathway, or short hairpin RNA targeting β‐catenin expression demonstrated that the invasive activity was not mediated by β‐catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context‐dependent. While Rspo2 and Wnt1 act synergistically in the β‐catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. J. Cell. Physiol. 227: 1960–1971, 2012.

Expression of Notch receptors, ligands and target genes during development of the mouse mammary gland

Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study, we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context. J. Cell. Physiol. 226: ??–??, 2011.

Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Recombinant R-spondin2 and Wnt3a Up- and Down- Regulate Novel Target Genes in C57MG Mouse Mammary Epithelial Cells

R-spondins (Rspos) comprise a family of four secreted proteins that have important roles in cell proliferation, cell fate determination and organogenesis. Rspos typically exert their effects by potentiating the Wnt/β-catenin signaling pathway. To systematically investigate the impact of Rspo/Wnt on gene expression, we performed a microarray analysis using C57MG mouse mammary epithelial cells treated with recombinant Rspo2 and/or Wnt3a. We observed the up- and down-regulation of several previously unidentified target genes, including ones that encode proteins involved in immune responses, effectors of other growth factor signaling pathways and transcription factors. Dozens of these changes were validated by quantitative real time RT-PCR. Time course experiments showed that Rspo2 typically had little or no effect on Wnt-dependent gene expression at 3 or 6 h, but enhanced expression at 24 h, consistent with biochemical data indicating that Rspo2 acts primarily to sustain rather than acutely increase Wnt pathway activation. Up-regulation of gene expression was inhibited by pre-treatment with Dickkopf1, a Wnt/β-catenin pathway antagonist, and by siRNA knockdown of β-catenin expression. While Dickkopf1 blocked Rspo2/Wnt3a-dependent down-regulation, a number of down-regulated genes were not affected by β-catenin knockdown, suggesting that in these instances down-regulation was mediated by a β-catenin-independent mechanism.

Cripto-1: an extracellular protein – connecting the sequestered biological dots

Abstract Cripto-1 (CR-1) is a multifunctional embryonic protein that is re-expressed during inflammation, wound repair, and malignant transformation. CR-1 can function either as a tethered co-receptor or shed as a free ligand underpinning its flexible role in cell physiology. CR-1 has been shown to mediate cell growth, migration, invasion, and induce epithelial to mesenchymal transition (EMT). The main signaling pathways mediating CR-1 effects include Nodal-dependent (Smad2/3) and Nodal-independent (Src/p44/42/Akt) signaling transduction pathways. In addition, there are several naturally occurring binding partner proteins (BPPs) for CR-1 that can either agonize or antagonize its bioactivity. We will review the collective role of CR-1 as an extracellular protein, discuss caveats to consider in developing a quantitation assay, define possible mechanistic avenues applicable for drug discovery, and report on our experimental approaches to overcome these problematic issues.

Abstract B27: Tackling mammary tumors by disrupting cancer stem cells via modulation of developmental signaling pathways

The master regulators of embryonic development, Cripto and Notch, play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells and in the adult organism particularly within the adult mammary stem cells, which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during mammary gland tumorigenesis to support the tumor-initiating cell/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of breast cancer. To investigate a possible interplay of Cripto and Notch pathways in mammary tumorigenesis and CSCs generation/maintenance, we developed a transgenic mouse model overexpressing Notch and lacking Cripto. We observed considerable tumor suppressor effects when one allele of Cripto was deleted, including significant reduction in the number of mammary hyperplasias, increased tumor latency, and the development of histologically well-differentiated tumors, preserving glandular-like mammary structures. Importantly, this phenotype could be associated with a putative role of Cripto in the specification of CSCs, observed by the lack of luminal progenitor markers, decreased tumor-spheres formation, less colonies in soft-agar, and a significantly reduced capacity to initiate mammary tumors in syngeneic FVB/N mice. Therefore, Cripto proved to contribute to Notch tumorigenesis and maintenance of CSCs, by preventing the differentiation of a parity-induced population of luminal progenitor cells, which is the target for Notch transformation. Searching for specific anti-Cripto-1 therapies that could be further translated to the clinic, we treated Notch-overexpressing/Cripto-wt animals with a synthetic anticancer agent, HKH40A, and observed a significant decrease in tumor formation/growth. The negative modulation of these pathways in the clinic may represent an important tool to prevent the generation and maintenance of CSCs, and preclude the initiation/promotion of breast cancer metastatic spread, resistance to therapy, and cancer recurrence. Citation Format: Maria Cristina Rangel, Ahmed Raafat, Nadia P. Castro, Malgorzata Klauzinska, Teresa Kosakowska-Cholody, Fariba Behbod, Stephen Lockett, Robert Callahan, Roger Chammas, David Salomon. Tackling mammary tumors by disrupting cancer stem cells via modulation of developmental signaling pathways [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B27.

Abstract LB-261: Mammary tumorigenesis in haploinsufficient Cripto-1 mice.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The embryonic gene Cripto-1 (Cr-1) is expressed at very low levels in normal adult tissues and detected at high levels in several human tumors. We have previously found that Cr-1 can facilitate the proteolytic intracellular processing of all four Notch receptors. Transgenic mice overexpressing the Notch4 intracellular domain (Int3) in the mammary gland under the control of the whey acidic protein (WAP) promoter rapidly develop mammary hyperplasias and undifferentiated mammary adenocarcinomas in aged nulliparous females and after multiple cycles of pregnancy. Eighty percent of WAP-Int3 females develop mammary tumors by the second pregnancy. To assess the contribution of Cr-1 in the hyperplasias and tumors that arise in transgenic WAP-Int3 mice, we developed a bi-transgenic mouse model lacking one allele of Cr-1, by crossing the knock-in Cr-1/LacZ mice (containing a promoter-less bacterial β-galactosidase gene which disrupts one of the Cr-1 alleles) to WAP-Int3 transgenic mice. We found a 90% reduction in the frequency of mammary hyperplasias in these Cr-1/LacZ/WAP-Int3 mice in comparison to WAP-Int3 mice. Additionally, in the absence of one allele of Cr-1, Int3-induced transformation of mouse mammary epithelial cells was delayed in vivo, and additional cycles of pregnancy were required to observe tumor growth. Notably, only 20% of the Int3/Cr-1 haploinsufficient mice developed mammary tumors by the second pregnancy, as compared to 80% of those mice that preserve two alleles of Cr-1. The few tumors derived from these mice also showed a significantly more differentiated histology in comparison to those tumors that arose from mice preserving both alleles of Cr-1. We are currently generating a mouse model which conditionally lacks the two alleles of Cr-1 in the mammary gland, what should better address our question regarding the functional significance of Cr-1 in the development of Int3-induced mammary tumorigenesis. Citation Format: Maria Cristina Rangel, Ahmed Raafat, Nadia P. Castro, Malgorzata Klauzinska, Hideaki Karasawa, Alyson Baker, Robert Callahan, David S. Salomon. Mammary tumorigenesis in haploinsufficient Cripto-1 mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2013-LB-261