Agr2 is a putative member of the protein disulfide isomerase family (Higa et al., 2011
; Park et al., 2009
; Zhao et al., 2010
) known to be up-regulated in a number of epithelial neoplasms including breast, prostate, pancreas, and ovarian cancers. In breast tumors its expression positively correlates with ER status (Barraclough et al., 2009
; Innes et al., 2006
; Maresh et al., 2010
; Park et al., 2011
; Ramachandran et al., 2008
; Vivekanandan et al., 2009
; Wu et al., 2008
; Zhang et al., 2005
). Functional studies involving cancer cell lines and engraftment experiments point to the involvement of Agr2 in a broad spectrum of pro-oncogenic features including promotion of proliferation, metastasis and decreased apoptosis (Vanderlaag et al., 2010
). Despite the presence of significant clinical data and suggestive in vitro
models, information on the role of Agr2 in normal mammary gland biology is lacking. Our study, introduces the first mammary gland specific deletion and overexpression Agr2 mouse models, demonstrating that Agr2 facilitates normal lobuloalveolar development in pregnancy through positive regulation of mammary epithelial cell proliferation. This work suggests that promotion of cell proliferation could be an early oncogenic feature of Agr2 overexpression.
Developmentally, the expression of Agr2
is low in virgin mammary glands, increasing during pregnancy and reaching its peak at lactation. Within 48 hours of weaning, Agr2
expression in the mammary gland decreases back to early pregnancy levels, suggesting a role during pregnancy and lactation. In contrast, Agr3
is minimally expressed in comparison to Agr2
throughout mammary gland development. While this suggests Agr3 may not play a prominent role in development, it has been reported to be overexpressed in ER-positive breast cacrcinomas (Fletcher et al., 2003
), and its function in normal mammary gland development and breast cancer remains to be elucidated.
To understand the role of Agr2 during post-natal mammary gland development we created mammary specific Agr2
knockout and overexpression mice. Consistent with low levels of Agr2
expression in virgin mice, Agr2
knockout virgin mice appear to undergo normal branching morphogenesis and elongation. However, Agr2
knockout pregnant mice (P12.5 and P17.5) have decreased lobuloalveolar mass in comparison to control animals, indicating a role for Agr2 in alveologenesis during pregnancy. In addition, we consistently observed a reduction in milk protein expression during late pregnancy and lactation in Agr2
knockout mice. Consistent with the pregnancy and lactational developmental defects in the Agr2
knockout mice, the rate of pup growth was significantly, although not dramatically, decreased after two weeks post-partum. One possible explanation for a moderate effect on pup growth is the short duration of weight monitoring experiments performed in this study. In addition, these moderate changes in weight could be due to the small litter sizes used in the experiments (5 pups each, the maximal litter size from knockout dams), and consequently greater overall amount of milk available per pup, providing nearly sufficient protein content for growth of pups from knockout dams. Also, other milk proteins and lipids may compensate for the decrease in casein and lactalbumin expression. For example, free fatty acid and triglyceride levels showed a trend toward increased levels in Agr2flox/floxMMTV-Cre mice
, although this did not reach statistical significance (Supplemental Figure 4
Interestingly, the gene expression pattern for milk proteins follows the expression pattern for Agr2 through pregnancy and lactation. This is especially the case for lactalbumin and Agr2, both of which are moderately expressed by mid-pregnancy, increase slightly by late-pregnancy, and increase sharply throughout lactation. Recently, it has been shown that AGR2 binds to nascent proteins and directs them to the endoplasmic reticulum lumen for folding and post-translational modifications (Higa et al., 2011
). We speculate that the tremendous amount of new milk proteins synthesized during lactation and the concurrent up-regulation of Agr2
during lactation may be in response to newly synthesized proteins as a part of the unfolded protein response machinery.
Consistent with Agr2 function in cell proliferation during pregnancy, induced overexpression of Agr2 caused increased branching and precocious lobuloalveolar development in virgin mice. This result is suggestive of a pro-oncogenic function of Agr2 since increased branching is often linked to pro-tumorigenic pathways (Fata et al., 2004
), possibly because the underlying mechanisms are partially shared with those causing pathological hyperplasia and invasiveness, features associated with pro-tumorigenic activity (Wang et al., 2007
). Moreover, Agr2 overexpression mice exhibit significantly increased proliferation, as measured by both BrdU incorporation and phospho-histone H3 staining. However, it is interesting to note that no tumors were observed during the study (the longest doxycycline treatment experiment lasted six months), indicating that Agr2
overexpression alone is either insufficient for tumorigenesis, requires longer induction, or plays a more prominent role in tumor progression and metastasis. Consistent with the overexpression, hyper-proliferative phenotype in hAgr2+
knockout pregnant mice exhibit a significantly reduced number of mitotic cells (as measured by pH3 staining), indicating that Agr2 physiologically regulates mammary epithelial cell proliferation.
In conjunction with our in vivo studies, we found that Agr2 is a regulator of cell proliferation in vitro. We demonstrate that Agr2 overexpression is sufficient to stimulate proliferation in ER-negative MCF10A cells. In ER-positive MCF7 cells stimulated with estradiol, Agr2 expression as well as overall cell proliferation is increased. Importantly, the estradiol-mediated increase in proliferation depends on Agr2 expression since knockdown of Agr2 significantly diminishes this effect.
Although the mechanisms by which Agr2 is involved in regulating proliferation are currently unknown, two recent studies provide potential clues. In an in vitro
system utilizing breast cancer cell lines, Vanderlaag and coworkers showed that CyclinD1 is downstream of Agr2 either directly or via positive regulation of ER-alpha (Vanderlaag et al., 2010
). Most recently, utilizing esophageal and lung adenocarcinoma cell lines, Dong et al. showed that Agr2 affects cell proliferation and anchorage independent growth in adenocarcinomas by regulating the expression of amphiregulin (Areg), a mitogenic ligand of the Epidermal Growth Factor Receptor (EGFR) pathway (Dong et al., 2011
). Both pathways implicated in these studies can be highly relevant to Agr2 induced proliferation in the normal mammary gland. For example, Sternlicht et al. has shown that Areg plays a prominent role during branching morphogenesis by activating EGFR signaling (Sternlicht et al., 2005
). While we show no effect of Agr2 deletion on branching morphogenesis, the induction of Areg
expression by Agr2 may be one possible mechanism by which Agr2 affects cell proliferation. We found Areg to be significantly down-regulated in the Agr2flox/floxMMTV-Cre
mammary gland at pregnancy day 12.5, but not day 17.5, and modestly decreased during lactation (Supplemental Figure 5A
). In contrast there was a significant increase of Areg
expression in the mammary glands of hAgr2+
mice and when Agr2
is overexpressed in MCF10A cells (Supplemental Figure 5
). However, knockdown of Agr2
expression in MCF7 cells showed no affect on Areg
expression (Supplemental Figure 5B
). Thus, Agr2 may in part influence cell proliferation by influencing Areg
expression and downstream EGFR signaling.
Estradiol/estrogen plays a critical role in proliferation of mammary epithelial cells during ductal elongation and branching as well as during lobuloalveolar development in pregnancy (Bocchinfuso et al., 2000
; Olsson et al., 1996
; Shyamala et al., 2002
; Zeps et al., 1998
). Anti-estrogens such as tamoxifen inhibit proliferation in ER-positive breast cancers by inhibiting the binding of estrogen to its receptor, affirming the role of estrogen during physiological as well as pathological proliferation in the mammary gland (Dorssers et al., 2001
expression is positively correlated with ER expression in breast cancer and Agr2
itself is an estradiol responsive gene (Innes et al., 2006
). Our data suggests that some of the effects of ER on proliferation in mammary gland are mediated through Agr2.
A recent study utilized ChIP-Seq to identify differential ER binding events in both good and poor prognosis ER-positive tumors, as well as metastases derived from ER-positive tumors (Ross-Innes et al., 2012
). Interrogating ER binding events reported in this study using a 20-kilobase window surrounding the Agr2
gene, an optimal size for searching for ER binding sites associated with target gene expression (Fullwood et al., 2009
), we located ER binding events near Agr2
in only one of the eight ER-positive tumors associated with good prognosis, while four of the seven poor prognosis tumors and all three of the metastases had multiple ER binding events surrounding the gene. These observations are consistent with previous studies linking increased Agr2
expression to poor prognosis in ER-positive tumors (Barraclough et al., 2009
; Hrstka et al., 2010
; Innes et al., 2006
), and suggest that Agr2 may be playing a more prominent role in difficult to treat ER-positive tumors that escape the current regimens of hormonal therapy. Interestingly, none of the ER-negative tumors used as controls in this study exhibited ER binding near the Agr2
gene, supporting previous experiments pointing to Agr2
as an ER-responsive gene.
We believe our study provides an important initial step in characterizing the role of Agr2 in normal mammary gland biology as a positive regulator of lobuloalveolar development in pregnancy and as a pro-proliferative factor in response to ER signaling. Furthermore, the knockout and overexpression models utilized in our study will serve as integral tools that will help elucidate the molecular mechanism by which Agr2 influences proliferation in both normal tissue homeostasis and cancer.