The Cain and Abl of epithelial-mesenchymal transition and transforming growth factor-β in mammary epithelial cells

Abstract

Transforming growth factor-β (TGF-β) normally inhibits breast cancer development by preventing mammary epithelial cell (MEC) proliferation, by inducing MEC apoptosis, and by creating cell microenvironments that maintain MEC homeostasis and prevent their uncontrolled growth and motility. Mammary tumorigenesis elicits dramatic alterations in MEC architecture and microenvironment integrity, which collectively counteract the tumor-suppressing activities of TGF-β and enable its stimulation of breast cancer invasion and metastasis. How malignant MECs overcome the cytostatic actions imposed by normal microenvironments and TGF-β, and how abnormal microenvironments conspire with TGF-β to stimulate the development and progression of mammary tumors remains largely undefined. These knowledge gaps have prevented science and medicine from implementing treatments effective in simultaneously targeting abnormal cellular microenvironments, and in antagonizing the oncogenic activities of TGF-β in developing and progressing breast cancers. c-Abl is a ubiquitously expressed nonreceptor protein tyrosine kinase that essentially oversees all aspects of cell physiology, including the regulation of cell proliferation, migration and adhesion, as well as that of cell survival. Thus, the biological functions of c-Abl are highly reminiscent of those attributed to TGF-β, including the ability to function as either a suppressor or promoter of tumorigenesis. Interestingly, while dysregulated Abl activity clearly promotes tumorigenesis in hematopoietic cells, an analogous role for c-Abl in regulating solid tumor development, including those of the breast, remains controversial. Here, we review the functions of c-Abl in regulating breast cancer development and progression, and in alleviating the oncogenic activities of TGF-β and its stimulation of epithelial-mesenchymal transition during mammary tumorigenesis.

Fig. 1

c-Abl antagonism fails to inhibit mammary tumor growth in mice. Female Balb/c mice were injected orthotopically with syngeneic 4T1 cells and treated daily beginning on day 8 after engraftment with either vehicle or imatinib (50 mg/kg/day) as indicated. Primary tumors were removed surgically and weighed at days 24 and 27 after engraftment. Bars indicate the mean tumor weight per group (6 mice/group). Reprinted with kind permission from Allington et al. [2009].

Fig. 2

Schematic depiction of the functional domains of human (1a and 1b) and murine (I and IV) Abl isoforms. The N terminus of c-Abl contains either an autoinhibitory Cap region or a consensus motif for myristoylation (black), which is followed by SH3 (blue; colors refer to the online version only) and SH2 (pink) domains, which is followed by the catalytic PTK domain (kinase, yellow). The central region of c-Abl possesses 4 PXXPXK/R sequences (purple), 3 nuclear localization sequences (NLS, white), and 3 high-mobility group-like boxes (HLB, green). Finally, the C terminus of c-Abl houses domains for binding globular (G, orange) and filamentous (F, gray) actin, as well as a nuclear export sequence (NES, brown). The oncogenic forms of Abl (v-Abl and BCR-Abl) contain modified N-terminal regions that disrupt the autoinhibitory functions normally mediated by the Cap region, which elicits constitutive PTK activity. The aberrant N terminus in v-Abl comprises a viral Gag sequence (light blue), while that in BCR-Abl comprises a portion of the N terminus of the BCR (red).

Fig. 3

Constitutive c-Abl activity suppresses EMT and induces MET in metastatic MECs. KD-Abl = Kinase-dead c-Abl mutant; shAbl = c-Abl deficient. a Direct FITC-conjugated phalloidin immunofluorescence was performed to monitor the actin cytoskeletal architecture in c-Abl-manipulated normal murine mammary gland (NMuMG) cells, which readily acquired mesenchymal morphologies in loss of c-Abl function MECs. b Bright-field images of c-Abl-manipulated 4T1 cells grown in traditional 2D tissue culture systems. Gain of c-Abl function elicited an apparent MET in 4T1 cells. c, d c-Abl-manipulated 4T1 cells were propagated for 7 days in compliant 3D organotypic cultures prior to analyzing their growth and morphology by capturing bright-field images (c), or by staining with FITC-conjugated phalloidin and DAPI (d). Gain of c-Abl function suppressed acinar growth and promoted normal acinar development, including partial hollowing of the resulting organoids. All are representative of 2–3 independent experiments and were reprinted with kind permission from Allington et al. [2009].

Fig. 4

Enforced c-Abl activation abrogates the growth of metastatic MECs in mice. a Female Balb/c mice were injected orthotopically with syngeneic parental (i.e. empty vector) or CST-Abl-expressing 4T1 cells (10,000 cells/animal; 12 animals/group), whose ability to grow as tumors was measured using digital calipers over a period of 30 days. Reprinted with kind permission from Allington et al. [2009]. b The mammary glands of mice injected with CST-Abl-expressing 4T1 cells were excised at day 51 after engraftment, and were subsequently dissociated enzymatically to produce a heterogeneous, single-cell suspension that was subjected to a clonogenic assay to reisolate reverted CST-Abl-expressing 4T1 cells.

Fig. 5

Schematic depiction of the role of c-Abl in suppressing EMT and oncogenic TGF-β signaling in normal and malignant MECs. In normal MECs, c-Abl activation (1) maintains adherens junction stability and cortical actin architecture; (2) mediates growth arrest in response to TGF-β and DNA damage; and (3) represses MMP expression and secretion. As developing mammary neoplasms become more and more malignant, oncogenic TGF-β signaling in conjunction with focal adhesion complex activation (e.g., β₃-integrin/focal adhesion kinase/Src) may promote the degradation of c-Abl and its uncoupling from the tumor-suppressing activities of TGF-β. The loss of c-Abl function ushers in the initiation of EMT and its dissolution of adherens junctions, its production and activation of matrix metalloproteinases, and its circumvention of cytostasis induced by TGF-β and DNA damage, which collectively enhance breast cancer progression and metastatic dissemination to distant locales. RI/RII = TGF-β type I receptor/TGF-β type II receptor; E-cad = E-cadherin; MDM2 = murine double minute 2.