gene is amplified in 15–30% of invasive breast cancers and this correlates with high grade tumors that are highly proliferative and convey poor outcome (Slamon et al., 1989
). Currently, the most effective therapy for this subtype of breast cancer is trastuzumab, a humanized monoclonal antibody that binds the extracellular domain of ErbB2 and inhibits receptor signaling. Although this therapy has proven successful for ~50% of patients with ErbB2-positive disease, many patients are resistant or develop resistance to this drug (Romond et al., 2005
; Piccart-Gebhart et al., 2005
). Furthermore, among those patients treated with trastuzumab, a significant proportion must discontinue treatment due to cardiotoxic side-effects (Seidman et al., 2002
). Besides the clinical limitations of trastuzumab, tumor studies in mice have revealed that recurrence occurs after complete suppression of ErbB2 even though it initiated the primary tumors (Moody et al., 2005
). This suggests that activation of additional autonomous oncogenic signals contributes to tumor cell survival and progression. Hence, identification of novel downstream targets of ErbB2 may be necessary to develop new treatment options.
ErbB2 is a member of the EGFR tyrosine kinase family, which also includes ErbB3 and ErbB4 (Yamamoto et al., 1986
). Although ErbB2 does not bind any known ligand, growth factor binding to other family members induces ErbB2 activity because it is the preferred heterodimerization partner (Graus-Porta et al., 1997
). Signaling via ErbB2 activates several major signaling pathways including MAPK (Ben Levy et al., 1994
), PI3K/AKT (Peles et al., 1992
), and PLCγ (Peles et al., 1991
). These pathways induce proliferation, in part by upregulating the cell cycle protein, Cyclin D1 (Lenferink et al., 2001
), which is essential for ErbB2-induced transformation (Yu et al., 2001
; Lee et al., 2000
). While several breakthroughs have been made in identifying early signaling events initiated by ErbB2, many of the downstream effectors of ErbB2-induced tumorigenesis remain unknown. Identification of these intermediates should provide significant insight into the molecular mechanisms employed by ErbB2 to induce breast cancer.
Correlative studies have indicated that the LIM-only protein 4 [LMO4] is overexpressed in the majority of breast cancers that also have ErbB2 amplification (Sum et al., 2005b
). LMO4 belongs to the LIM-only family, which is characterized by two tandem, non-DNA binding LIM domains that confer the potential to interact with multiple proteins simultaneously (Rabbitts, 1998
). Because LMO proteins bind to transcription factors and cofactors, they have been implicated in the control of gene expression by forming multimeric transcriptional complexes (Sum et al., 2002
Conditional loss of LMO4
in the mammary gland results in reduced lobuloalveolar development with a concomitant decrease in cellular proliferation (Sum et al., 2005c
; Wang et al., 2007
). These findings suggest that LMO4 is necessary for expansion of the epithelial compartment of the mammary gland, possibly due to its regulation of proliferation. Supporting this notion, targeted overexpression of LMO4 in mouse mammary glands induces hyperplasia and tumors (Sum et al., 2005b
). In vitro
, overexpression of LMO4 or its binding partner Ldb1, inhibited expression of differentiation markers in mammary epithelial cells (Visvader et al., 2001
), while LMO4 silencing is associated with decreased growth, migration and invasion of breast cancer cells (Sum et al., 2005b
). Although these data implicate LMO4 in the pathogenesis of breast cancer through its modulation of proliferation, the mechanisms by which LMO4 regulates proliferation and the processes that control LMO4 expression and activity remain unknown.
In human breast cancers, LMO4 is overexpressed in 56% of all breast tumors (Visvader et al., 2001
), and 65% of those that are positive for ErbB2 (Sum et al., 2005b
mRNA is also elevated in several other cancers including prostate cancers (Mousses et al., 2002
) and small cell lung carcinomas that fail chemotherapy (Taniwaki et al., 2006
). Most importantly, multivariate analysis of human breast cancers suggests that LMO4 overexpression may account for much of the aggressiveness associated with ErbB2-overexpression (Sum et al., 2005b
). However, a functional link between LMO4 and ErbB2 has yet to be identified.
Herein, we report that ErbB2 and its downstream signaling protein, PI3K, regulate LMO4 expression in breast cancer cells. Furthermore, LMO4 is necessary for ErbB2-mediated proliferation and cell cycle progression. LMO4 expression fluctuates throughout the cell cycle and is required for sustained expression of key cell cycle regulators: the G1 cyclin, Cyclin D1, and the G2/M ubiquitin E3-ligase component, cullin-3. We conclude LMO4 is an essential downstream effector of ErbB2-induced cell cycle progression and likely plays an important role in modulating the aggressiveness of various types of breast cancer.