Regulation of transcription factor and cofactor activity by subcellular compartmentalization is well documented [32
]. A common mechanism is sequestration of the factor into inactive compartments, and this typically takes place via direct or indirect association with the cytoskeleton [35
]. LRP16 is a new type of ERα coactivator that augments the receptor's transcriptional activity in a ligand-dependent manner and can have a profound impact on the final output of cellular signaling [19
]. LRP16 is though to modulate ERα activity in the nucleus; in the present paper we have confirmed that a LRP16-GFP fusion protein localizes primarily to MCF-7 cell nuclei. We also report a new LRP16 ligand, K18, identified by yeast two-hybrid screening. K18 is a member of the family of intermediate filament proteins that contribute to cytoskeletal architecture. In the present study we report that K18 binds to and sequesters LRP16 in the cytoplasm, thus preventing its nuclear action and attenuating both E2-induction of ERα target genes and E2-stimulated cell cycle progression of MCF-7 cells. These findings underscore the functional role of K18 in regulating the ERα signaling pathway.
LRP16, a member of the macro domain protein superfamily, contains a single stand-alone macro module in its C-terminal region [14
]. We recently demonstrated that LRP16 is a non-redundant coactivator of both ERα and AR [15
]. LRP16 was also able to interact with another 4 nuclear receptors (NRs) in vitro
, including estrogen receptor β(ERβ), the glucocorticoid receptor, and peroxisome proliferator-activated receptors α and γ, and can efficiently amplify the transactivation of these NRs in a ligand-dependent manner [15
]. Our finding that K18 binds to and sequesters LRP16 in the cytoplasm suggests that differential tissue expression of K18 could constitute a new layer in the regulatory cascade of signaling pathways in which LRP16 participates.
Keratins (KRTs) provide mechanical stability to tissues, as evidenced by the range of pathological phenotypes seen in patients bearing mutations in epidermal keratins [39
]. The intermediate filament network in simple glandular epithelial cells predominantly consists of heterotypic complexes of KRTs K8 and K18. Additional evidence for a more widespread role of KRTs comes from mouse gene knockout studies. Double deletion of the genes encoding K18 and K19 results in complete loss of a functional cytokeratin skeleton and embryonic lethality [40
]. The assembly of intermediate filament involves several steps during which the α-helical rod domain of the cytokeratin molecules plays a central role [41
]. The head and the tail domains are not thought to be part of the filamentous backbone, and instead these protrude laterally and contribute to profilament and intermediate filament packing and to intermediate filament interaction with other cellular components [44
]. By associating with signal transduction factors K18 may modulate both intracellular signaling and gene transcription. For example, K18 is known to bind specifically to the tumor necrosis factor (TNF) receptor type 1(TNFR1)-associated death domain protein (TRADD) through its N-terminal region and prevent TRADD from binding to activated TNFR1, thus attenuating TNF-induced apoptosis in simple epithelial cells [44
We report here that K18 binding to LRP16 is primarily mediated by the C-terminal region of K18 and the single macro domain of LRP16. We used two independent approaches, including subcellular localization analysis of GFP-tagged LRP16 and cytoplasmic/nuclear LRP16 protein expression analyses, to demonstrate that ectopic K18 expression in MCF-7 cells sequesters LRP16 in the cytoplasm. Conversely, knockdown of K18 gene expression increased the nuclear localization of LRP16. By binding to and sequestering LRP16 in the cytoplasm, K18 prevents the nuclear action of LRP16 and attenuates ERα signaling, thus blunting estrogen-stimulated cell-cycle progression of ERα-positive breast cancer cells.
Accumulating evidence from clinicopathological observations has shown that the level of K18
gene expression correlates inversely with the progression of breast cancer [25
]. Several reports have proposed that downregulation of K18 might increase the invasiveness of breast cancer cells [25
]. It was previously demonstrated that overexpression of K18 in the ERα-negative and highly invasive MDA-MB-231 breast cancer cell line caused a marked reduction in the aggressiveness of the cells in vitro
and in vivo
but had no significant effect on cell growth rate. This change was accompanied by complete loss of the previously strong vimentin expression in the parent cell line and upregulation of adhesion proteins such as E-cadherin [31
]. However, experimental studies and clinicopathological observations also revealed a significant association between K18 expression and the proliferation rate of breast cancer cells. Analysis of the association between K18 expression and different clinicopathological risk factors revealed that K18 expression is highly and significantly correlated with size (pT1-3), differentiation grade, and mitotic index of the primary tumor [27
]. These parameters are a function of the proliferation rate of the primary tumor, and this suggests that there is a relationship between downregulation of K18 expression and increased proliferative activity. In addition, the expression of the proliferation-associated antigen Ki-67 is significantly associated with the downregulation of K18 in a subset of primary breast carcinomas [27
]. Moreover, cell culture experiments on bone-marrow micrometastases of breast cancer have indicated that most proliferating tumor cells lack detectable expression of K18 protein [28
]. These previous data suggested that K18 might make an important contribution to tumor metastasis as well as to tumor cell growth. In the present study we have demonstrated that, by blunting estrogen-stimulated ERα signaling activity, K18 can significantly suppress the growth response of MCF-7 cells to estrogen. We propose that the regulatory mechanism of ERα transactivation by the K18-LRP16 association might explain in part the relationship between K18 downregulation and increased proliferative activity of breast cancers. However, K18 loss is also associated with the metastasis of ERα-negative breast cancers (47), and it therefore appears likely that K18 can modulate breast cancer progression by more than one mechanism.
Oncogenesis in breast cancer commonly involves excess activation of ERα signaling. We previously reported that LRP16 mRNA is overexpressed in nearly 40% of all primary breast cancer samples [21
]. LRP16 overexpression in breast cancer cells is tightly linked with cell proliferation and enhanced ERα activation [16
]. As a functional suppressor of LRP16, K18 is frequently absent from different types of breast carcinoma [25
]. Excess activation of ERα function in tumor cells is commonly mediated by overexpression of ERα and/or its coactivators including LRP16 [6
]. We now propose a further level of regulation that can modulate ERα function in breast cancer. Loss of K18 from ERα-positive breast tumor cells releases the functional activity of LRP16, and is thus likely to promote tumor cell proliferation. Tests that evaluate the subcellular localization of LRP16 in ERα-positive breast tumor cells therefore have potential in the categorization of different clinopathological stages.