Jenifer R. Prosperi

A Wnt-ow of Opportunity: Targeting the Wnt/β-Catenin Pathway in Breast Cancer

Aberrant activation of the Wnt/beta-catenin signaling pathway is a hallmark of many tumors, including breast cancer. In the normal breast, tightly regulated expression of Wnt/beta-catenin pathway components, including Wnts and the APC tumor suppressor, dictates its role in balancing stem cell self-renewal, maintenance and differentiation during embryonic and postnatal development. Therefore, not surprisingly, dysregulation of Wnt/beta-catenin signaling through overexpression of pathway activators, such as Wnts or stabilized beta-catenin, or targeted disruption of inhibitors, such as APC, leads to mammary tumorigenesis in several genetically engineered mouse (GEM) models. These models are powerful tools to dissect the importance of Wnt/beta-catenin signaling in human breast cancer because they recapitulate some of the histological features of human breast cancers that demonstrate pathway dysregulation. Over the last decade, numerous approaches have been developed to target the Wnt/beta-catenin pathway in tumor cells, from antagonizing Wnt ligand secretion or binding to promoting beta-catenin degradation to specifically blocking beta-catenin-mediated transcriptional activity. Despite sizeable hurdles because of gaps in our knowledge of most efficacious ways to inhibit the pathway, the breast cancer subtypes to target and how pathway antagonists might be used in combination therapy, crippling Wnt/beta-catenin signaling offers a tremendous opportunity to impact breast cancer pathogenesis. This review will provide an overview of the current understanding of Wnt/beta-catenin pathway involvement in regulating normal breast development and morphogenesis, the generation of Wnt/beta-catenin-dependent GEM models of human breast cancer, upregulation of signaling in human breast cancers and the compelling therapeutic strategies aimed at targeting the Wnt/beta-catenin pathway that show promising anti-tumor activity.

β-Catenin Is Required for the Tumorigenic Behavior of Triple-Negative Breast Cancer Cells

Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether β-catenin was necessary for tumorigenic behaviors in vivo and in vitro. β-catenin expression level was significantly reduced in two human triple-negative breast cancer cell lines, MDA-MB-231 and HCC38, using lentiviral delivery of β-catenin-specific small hairpin RNAs (shRNAs). Upon implantation of the cells in the mammary fat pad of immunocompromised mice, we found that β-catenin shRNA HCC38 cells formed markedly smaller tumors than control cells and grew much more slowly. In in vitro assays, β-catenin silencing significantly reduced the percentage of Aldefluor-positive cells, a read-out of the stem-like cell population, as well as the expression of stem cell-related target genes including Bmi-1 and c-Myc. β-catenin-knockdown cells were also significantly impaired in their ability to migrate in wound-filling assays and form anchorage-independent colonies in soft agar. β-catenin-knockdown cells were more sensitive to chemotherapeutic agents doxorubicin and cisplatin. Collectively, these data suggest that β-catenin is required for triple-negative breast cancer development by controlling numerous tumor-associated properties, such as migration, stemness, anchorage-independent growth and chemosensitivity.

The APC tumor suppressor is required for epithelial integrity in the mouse mammary gland.

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor has been associated with mammary tumorigenesis in mouse models and through epidemiological studies of human breast cancers, but the normal role for APC in mammary development has not been thoroughly characterized. We report here that ApcMin/+ mice containing one functional allele of Apc have severely disrupted lobuloalveolar development during pregnancy and lactation, time points at which Apc gene expression is at its highest levels in normal mice. This phenotype was accompanied by altered proliferation during pregnancy and involution, increased apoptosis throughout lactation, the formation of preneoplastic lesions and changes in specific genes associated with each of these processes. Neither modifications in β‐catenin localization, nor the expression of β‐catenin transcriptional target genes, were observed in ApcMin/+ mammary tissues; however, tissues from lactating ApcMin/+ mice had a significantly altered epithelial architecture, including disrupted localization of junctional proteins and polarization. Consistent with these findings, APC knockdown in non‐transformed mouse mammary epithelial cells in vitro resulted in altered monolayer formation and proliferation without changes in β‐catenin‐mediated transcription. These results suggest that APC expression is tightly regulated during mammary gland development and is required for normal mammary homeostasis and tumor suppression primarily through maintaining epithelial integrity. J. Cell. Physiol. 220: 319–331, 2009.

Application of physicochemically modified silicon substrates as reverse phase protein microarrays

Physicochemically modified silicon substrates can provide a high quality alternative to nitrocellulose-coated glass slides for use in reverse-phase protein microarrays. Enhancement of protein microarray sensitivities is an important goal, especially because molecular targets within patient tissues exist in low abundance. The ideal array substrate has a high protein binding affinity and low intrinsic background signal. Silicon, which has low intrinsic autofluorescence, is being explored as a potential microarray surface. In a previous paper ( Nijdam , A. J. ; Cheng , M. M.-C. ; Fedele , R. ; Geho , D. H. ; Herrmann , P. ; Killian , K. ; Espina , V. ; Petricoin , E. F. ; Liotta , L. A. ; Ferrari , M. Physicochemically Modified Silicon as Substrate for Protein Microarrays . Biomaterials 2007 , 28 , 550 - 558 ), it is shown that physicochemical modification of silicon substrates increases the binding of protein to silicon to a level comparable with that of nitrocellulose. Here, we apply such substrates in a reverse-phase protein microarray setting in two model systems.

Apc Mutation Enhances PyMT-Induced Mammary Tumorigenesis

The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in up to 70% of human breast cancers. In mouse models, Apc mutation disrupts normal mammary development and predisposes to mammary tumor formation; however, the cooperation between APC and other mutations in breast tumorigenesis has not been studied. To test the hypothesis that loss of one copy of APC promotes oncogene-mediated mammary tumorigenesis, ApcMin/+ mice were crossed with the mouse mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) or MMTV-c-Neu transgenic mice. In the PyMT tumor model, the ApcMin/+ mutation significantly decreased survival and tumor latency, promoted a squamous adenocarcinoma phenotype, and enhanced tumor cell proliferation. In tumor-derived cell lines, the proliferative advantage was a result of increased FAK, Src and JNK signaling. These effects were specific to the PyMT model, as no changes were observed in MMTV-c-Neu mice carrying the ApcMin/+ mutation. Our data indicate that heterozygosity of Apc enhances tumor development in an oncogene-specific manner, providing evidence that APC-dependent pathways may be valuable therapeutic targets in breast cancer. Moreover, these preclinical model systems offer a platform for dissection of the molecular mechanisms by which APC mutation enhances breast carcinogenesis, such as altered FAK/Src/JNK signaling.

APC selectively mediates response to chemotherapeutic agents in breast cancer

BackgroundThe Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70 % of sporadic breast cancers depending on subtype; however, the effects of APC mutation on tumorigenic properties remain unexplored. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we identified enhanced breast tumorigenesis and alterations in genes critical in therapeutic resistance independent of Wnt/β-catenin signaling. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. Mechanistic studies in tumor-derived cell lines demonstrated that focal adhesion kinase (FAK)/Src/JNK signaling regulated the enhanced proliferation downstream of Apc mutation. Despite this mechanistic information, the role of APC in mediating breast cancer chemotherapeutic resistance is currently unknown.MethodsWe have examined the effect of Apc loss in MMTV-PyMT mouse breast cancer cells on gene expression changes of ATP-binding cassette transporters and immunofluorescence to determine proliferative and apoptotic response of cells to cisplatin, doxorubicin and paclitaxel. Furthermore we determined the added effect of Src or JNK inhibition by PP2 and SP600125, respectively, on chemotherapeutic response. We also used the Aldefluor assay to measure the population of tumor initiating cells. Lastly, we measured the apoptotic and proliferative response to APC knockdown in MDA-MB-157 human breast cancer cells after chemotherapeutic treatment.ResultsCells obtained from MMTV-PyMT;ApcMin/+ tumors express increased MDR1 (multidrug resistance protein 1), which is augmented by treatment with paclitaxel or doxorubicin. Furthermore MMTV-PyMT;ApcMin/+ cells are more resistant to cisplatin and doxorubicin-induced apoptosis, and show a larger population of ALDH positive cells. In the human metaplastic breast cancer cell line MDA-MB-157, APC knockdown led to paclitaxel and cisplatin resistance.ConclusionsAPC loss-of-function significantly increases resistance to cisplatin-mediated apoptosis in both MDA-MB-157 and the PyMT derived cells. We also demonstrated that cisplatin in combination with PP2 or SP600125 could be clinically beneficial, as inhibition of Src or JNK in an APC-mutant breast cancer patient may alleviate the resistance induced by mutant APC.

Dysregulation of the wnt pathway in solid tumors

Activation of the WNT signaling pathway has long been implicated in driving cancer pathogenesis from compelling evidence derived from complementary in vitro and animal studies. Moreover, there are extensive data supporting the nuclear localization of β-catenin, a surrogate marker frequently used as a read-out of WNT pathway activation, in addition to the overexpression of pathway activators and loss of pathway inhibitors in human cancers. Despite being most often linked with colorectal cancer, WNT pathway activation has now been associated with virtually every type of solid cancer that occurs in humans, although the frequency can vary dramatically among tumor subtypes and specific pathway components. These findings have significant implications regarding the mechanisms by which pathway activation might drive tumor development in specific tumor subtypes as well as the potential utility and impact of targeting this pathway therapeutically. In this chapter, we will summarize, both by tumor type and pathway components, the expansive evidence suggesting that the pathway is dysregulated in nearly half of all the most common types of human malignancies.

The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis.

The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further supports the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis.

APC loss in breast cancer leads to doxorubicin resistance via STAT3 activation

Resistance to chemotherapy is one of the leading causes of death from breast cancer. We recently established that loss of Adenomatous Polyposis Coli (APC) in the Mouse Mammary Tumor Virus – Polyoma middle T (MMTV-PyMT) transgenic mouse model results in resistance to cisplatin or doxorubicin-induced apoptosis. Herein, we aim to establish the mechanism that is responsible for APC-mediated chemotherapeutic resistance. Our data demonstrate that MMTV-PyMT;ApcMin/+ cells have increased signal transducer and activator of transcription 3 (STAT3) activation. STAT3 can be constitutively activated in breast cancer, maintains the tumor initiating cell (TIC) population, and upregulates multidrug resistance protein 1 (MDR1). The activation of STAT3 in the MMTV-PyMT;ApcMin/+ model is independent of interleukin 6 (IL-6); however, enhanced EGFR expression in the MMTV-PyMT;ApcMin/+ cells may be responsible for the increased STAT3 activation. Inhibiting STAT3 with a small molecule inhibitor A69 in combination with doxorubicin, but not cisplatin, restores drug sensitivity. A69 also decreases doxorubicin enhanced MDR1 gene expression and the TIC population enhanced by loss of APC. In summary, these results have revealed the molecular mechanisms of APC loss in breast cancer that can guide future treatment plans to counteract chemotherapeutic resistance.

Epithelial Membrane Protein 2 and β1 integrin signaling regulate APC-mediated processes

Abstract Adenomatous Polyposis Coli (APC) plays a critical role in cell motility, maintenance of apical‐basal polarity, and epithelial morphogenesis. We previously demonstrated that APC loss in Madin Darby Canine Kidney (MDCK) cells increases cyst size and inverts polarity independent of Wnt signaling, and upregulates the tetraspan protein, Epithelial Membrane Protein 2 (EMP2). Herein, we show that APC loss increases &bgr;1 integrin expression and migration of MDCK cells. Through 3D in vitro model systems and 2D migration analysis, we have depicted the molecular mechanism(s) by which APC influences polarity and cell motility. EMP2 knockdown in APC shRNA cells revealed that APC regulates apical‐basal polarity and cyst size through EMP2. Chemical inhibition of &bgr;1 integrin and its signaling components, FAK and Src, indicated that APC controls cyst size and migration, but not polarity, through &bgr;1 integrin and its downstream targets. Combined, the current studies have identified two distinct and novel mechanisms required for APC to regulate polarity, cyst size, and cell migration independent of Wnt signaling. Highlights&bgr;1 integrin is upregulated in APC knockdown MDCK cells.APC loss increases cell motility during wound‐healing assays.EMP2 regulates APC‐mediated cyst size and apical polarity, but has no impact on migration.&bgr;1 integrin signaling mediates cyst size and migration due to APC loss, with no effect on polarity.