Kathleen H. Goss

Biology of the Adenomatous Polyposis Coli Tumor Suppressor

The adenomatous polyposis coli (APC) gene was first identified as the gene mutated in an inherited syndrome of colon cancer predisposition known as familial adenomatous polyposis coli (FAP). Mutation of APC is also found in 80% of all colorectal adenomas and carcinomas and is one of the earliest mutations in colon cancer progression. Similar to other tumor suppressor genes, both APC alleles are inactivated by mutation in colon tumors, resulting in the loss of full-length protein in tumor cells. The functional significance of altering APC is the dysregulation of several physiologic processes that govern colonic epithelial cell homeostasis, which include cell cycle progression, migration, differentiation, and apoptosis. Roles for APC in some of these processes are in large part attributable to its ability to regulate cytosolic levels of the signaling molecule beta-catenin and to affect the transcriptional profile in cells. This article summarizes numerous genetic, biochemical, and cell biologic studies on the mechanisms of APC-mediated tumor suppression. Mouse models of FAP, in which the APC gene has been genetically inactivated, have been particularly useful in testing therapeutic and chemopreventive strategies. These data have significant implications for colorectal cancer treatment approaches as well as for understanding other disease genes and cancers of other tissue types.

Wnt/β-Catenin Pathway Activation Is Enriched in Basal-Like Breast Cancers and Predicts Poor Outcome

Although Wnt/beta-catenin pathway activation has been implicated in mouse models of breast cancer, there is contradictory evidence regarding its importance in human breast cancer. In this study, invasive and in situ breast cancer tissue microarrays containing luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)(+)/ER(-) and basal-like breast cancers were analyzed for beta-catenin subcellular localization. We demonstrate that nuclear and cytosolic accumulation of beta-catenin, a read-out of Wnt pathway activation, was enriched in basal-like breast cancers. In contrast, membrane-associated beta-catenin was observed in all breast cancer subtypes, and its expression decreased with tumor progression. Moreover, nuclear and cytosolic localization of beta-catenin was associated with other markers of the basal-like phenotype, including nuclear hormone receptor and HER2 negativity, cytokeratin 5/6 and vimentin expression, and stem cell enrichment. Importantly, this subcellular localization of beta-catenin was associated with a poor outcome and is more frequently observed in tumors from black patients. In addition, beta-catenin accumulation was more often observed in basal-like in situ carcinomas than other in situ subtypes, suggesting that activation of this pathway might be an early event in basal-like tumor development. Collectively, these data indicate that Wnt/beta-catenin activation is an important feature of basal-like breast cancers and is predictive of worse overall survival, suggesting that it may be an attractive pharmacological target for this aggressive breast cancer subtype.

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.

Attenuated APC alleles produce functional protein from internal translation initiation

Some truncating mutations of the APC tumor suppressor gene are associated with an attenuated phenotype of familial adenomatous polyposis coli (AAPC). This work demonstrates that APC alleles with 5′ mutations produce APC protein that down-regulates β-catenin, inhibits β-catenin/T cell factor-mediated transactivation, and induces cell-cycle arrest. Transfection studies demonstrate that cap-independent translation is initiated internally at an AUG at codon 184 of APC. Furthermore, APC coding sequence between AAPC mutations and AUG 184 permits internal ribosome entry in a bicistronic vector. These data suggest that AAPC alleles in vivo may produce functional APC by internal initiation and establish a functional correlation between 5′ APC mutations and their associated clinical phenotype.

Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/β-Catenin Signaling

Wnt/β-catenin signal transduction requires direct binding of β-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. β-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-β-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/β-catenin in breast cancer.

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.

β-catenin regulates c-Myc and CDKN1A expression in breast cancer cells

We previously reported that the Wnt pathway is preferentially activated in basal‐like breast cancer. However, the mechanisms by which the Wnt pathway regulates down‐stream targets in basal‐like breast cancer, and the biological significance of this regulation, are poorly understood. In this study, we found that c‐Myc is highly expressed in the basal‐like subtype by microarray analyses and immunohistochemical staining. After silencing β‐catenin using siRNA, c‐Myc expression was decreased in non‐basal‐like breast cancer cells. In contrast, c‐Myc mRNA and protein expression were up‐regulated in the basal‐like breast cancer cell lines. Decreased c‐Myc promoter activity was observed after inhibiting β‐catenin by siRNA in non‐basal‐like breast cancer cells; however, inhibition of β‐catenin or over‐expression of dominant‐negative LEF1 had no effect on c‐Myc promoter activity in basal‐like breast cancer cell lines. In addition, CDKN1A mRNA and p21 protein expression were significantly increased in all breast cancer cell lines upon β‐catenin silencing. Interestingly, inhibiting β‐catenin expression alone did not induce apoptosis in breast cancer cell lines despite c‐Myc regulation, but we observed a modest increase of cells in the G1 phase of the cell cycle and decrease of cells in S phase upon β‐catenin silencing. Our findings suggest that the regulation of c‐Myc in breast cancer cells is dependent on the molecular subtype, and that β‐catenin‐mediated regulation of c‐Myc and p21 may control the balance of cell death and proliferation in breast cancer.

APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology

BackgroundThe APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation.MethodsThe localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity.ResultsAPC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton.ConclusionsThese findings indicate that membrane protrusions with APC/β-catenin-containing puncta control the migratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later stages of tumor progression might impact tumor cell dissemination or colonization.

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.