Primera infancia, infancia y adolescencia

Several regulators and potential substrates for AGR2 highlighted so far are known to play a role in tumourigenesis, and AGR2 itself has been associated with the progression of a number of different cancer types, although hormone-dependent cancers such as breast and prostate cancers have been the most studied, given the regulation of AGR2 by oestrogens and androgens (see above).

1.4.3.1 Hormone-dependent cancers

AGR2 seems to be particularly associated with the progression of breast cancer, possibly due to its role in breast tissue development, where it mediates proliferation of the mammary epithelium [456]. Multiple reports have identified its expression as a marker of disease stage and as a prognostic factor for patient survival in breast cancer, where increased expression of AGR2 is associated with reduced patient survival [309, 443, 457- 462]. Importantly, AGR2 is also implicated in the resistance of breast cancers to drug treatment. AGR2 was shown to mediate resistance to cisplatin [463], where tumour xenografts stably-expressing AGR2 were insensitive to cisplatin-mediated growth arrest, in contrast to isogenic xenografts not expressing AGR2. Tamoxifen treatment leads to upregulation of AGR2 expression, directly through an OR and Akt-dependent manner [459, 460], but also in an OR-independent fashion through Foxa1 [393]. Thus, AGR2 expression is also predictive of Tamoxifen resistance [460]. On the other hand, treatment of patients with aromatase inhibitors such as letrozole and anastrozole downregulates the expression of AGR2 [464] (presumably through loss of oestrogen-mediated gene transcription) and in one patient cohort, those who responded the best to letrozole treatment corresponded to those patients that saw the largest suppression of AGR2 expression [459]. Therefore combination therapies including drugs affecting AGR2 expression might have a favourable outcome for breast cancer patients.

A similar role for AGR2 in the progression of prostate cancer is also apparent, with a number of studies again showing a correlation between AGR2 and the occurrence of prostate cancer, as well as decreased patient survival [388, 391, 444, 465-469]. Interestingly, studies in prostate cancer cells have revealed that knockdown of AGR2 can promote cellular senescence [470], but also that ErbB3 binding protein 1 (EBP1) acts as a negative regulator of AGR2 expression [305]. EBP1 is often downregulated in prostate

Chapter 1 Introduction

32 cancers [471, 472], and seems to inhibit AGR2 expression through inhibition of Foxa1 and 2. As downregulation of EBP1 is associated with resistance to hormone treatment [471] and is also presumably associated with upregulation of AGR2 expression [305], AGR2 could be a valid target for reducing hormone treatment resistance in prostate cancer, in a similar fashion to that seen in breast cancer.

The presence of AGR2 acts as a similar prognostic factor for other hormone- dependent cancers. It is a marker of higher grade ovarian cancers, and can also be used to distinguish between serous and mucinous-type ovarian cancers [301, 473-476]. Pancreatic cancers are also sensitive to oestrogens [477], and the expression of AGR2 also appears to correlate with appearance of neoplastic or pre-neoplastic pancreatic cells [302, 412, 433, 437, 440, 478-480].

The prevalence of elevated AGR2 levels in these cancers (but also in some non- hormone-dependent cancers, see below) may relate to its pro-proliferative and anti- apoptotic effects. AGR2 mediates growth-regulatory effects in several cancer cell lines [301, 393, 440, 459], where AGR2 knockdown results in decreased or inhibited growth. More strikingly, AGR2 knockdown also results in apoptosis of several cancer cell lines [415, 445, 459, 470, 481] but in the context of breast cancers at least, this only appears to be true for OR-positive cell lines [482]. This suggests that some cancers may be dependent on AGR2 for survival. AGR2 appears to mediate these effects, at least in part, through upregulation of the cell cycle protein cyclin D1, as well as the pro-survival protein survivin [482], although

how it influences the cellular abundance of these proteins is not clear. Additionally, AGR2 is also an inhibitor of p53 activity, preventing activation of pro-apoptotic protein transcription through inhibiting its phosphorylation [438, 445]. Again, whether these effects are mediated through direct interactions or are the result of influences on upstream regulators is unknown.

1.4.3.2 Non-hormone dependent cancers

The occurrence of AGR2 expression in non-hormone dependent cancers is not as widely reported, presumably due to the strong effects of oestrogens and androgens on AGR2 expression. Nonetheless, AGR2 also appears to play a role in carcinogenesis in other tissues, generally those that contain a secretory epithelium.

AGR2 has been associated with the progression of oesophageal carcinomas, where it tends to be upregulated relative to normal tissue [394, 419, 445, 481, 483-485]. As

Chapter 1 Introduction

33 oesophageal carcinomas have been reported to display a characteristic inhibition of p53 [486], AGR2 may promote disease progression through inhibition of p53 activation [445]. Similarly, there is a correlation between AGR2 expression and disease progression in lung cancers [303, 487-491].

In cancers originating from the liver, the role of AGR2 appears to depend on the specific sub-type of carcinoma. As mentioned previously, AGR2 is expressed during liver and biliary tree morphogenesis [403] and seems to be maintained in normal liver [492], as well as in extrahepatic and some intrahepatic cholangiocarcinomas, where AGR2 expression correlates with mucus production [403]. AGR2 was similarly overexpressed in fibrolamellar carcinomas, but not in primary hepatocellular carcinomas (HCC), or at least only very rarely [403, 492]. However, high levels of AGR2 were found in metastatic HCC cells [304], suggesting that different selection pressures exist on AGR2 expression, depending on the cell type of origin. Furthermore, Yu and colleagues demonstrated that AGR2 directly interacted with elements of the MAPK and caspase pathways using co-immunoprecipitation experiments, and that this may contribute to the induction of metastasis in these cells [304]. However, the cellular expression levels of these potential interacting proteins were not investigated, so it is unclear if AGR2 affects the abundance of these proteins (in a similar way to mucins) or whether interaction with AGR2 results in some activation or inhibitory action. Any activity not based on protein abundance suggests an additional, chaperone-independent role for AGR2. Likewise, in colorectal carcinomas, where AGR2 expression also correlates with mucus production and can distinguish between mucinous and non-mucinous carcinomas, elevated expression of AGR2 can predict both better and poorer patient outcome, based on the specific cancer sub-type [493-495]. However, it is possible that the increase in serum AGR2 mRNA in colorectal cancer patients identified by Valladares-Ayerbes and colleagues [496] could also reflect the expression of AGR2 in circulating tumour cells as reported previously [306], thus again implicating AGR2 in the metastatic process.

1.4.3.3 AGR2 downregulation in cancer

Whilst the majority of reports demonstrate AGR2 expression to be an indicator of poor patient outcome, a number of studies have also demonstrated a correlation between high levels of AGR2 expression and better disease prognosis. Two individual studies of breast cancer and prostate cancer showed that AGR2 expression correlated with improved

Chapter 1 Introduction

34 patient survival [497, 498]. However, this is not entirely unexpected, given the role of AGR2 in differentiation of breast, intestinal and stomach tissues [409, 422, 424, 439, 456], where in the latter, loss of AGR2 leads to dedifferentiation and metaplasia [409, 439]. It is also important to note that the aforementioned studies on breast and prostate cancers did not stratify groups based on hormone receptor status. The fact that hormone receptor positive cancers are generally more treatable than receptor-negative cancers [499], and that AGR2 expression correlates with hormone receptor expression, may explain the apparent protective effect of AGR2. There are similar reports of AGR2-protective effects in ovarian cancer and colorectal cancer [493, 500], but this also positively correlates with cell differentiation in ovarian cancer [500] and probably also in colorectal cancer, given the role of AGR2 in the development and maintenance of the intestinal epithelium (see Section 1.4.2.1). Also in biliary tract tumours, expression of AGR2 correlates with cell differentiation, such that AGR2 can be downregulated during progression of these tumours [501].It appears then that, in tissues where AGR2 is normally expressed and functions normally, AGR2 tends to be lost during tumourigenesis due to the loss of a differentiated cell phenotype, whereas in tissues where AGR2 is not normally, or only weakly expressed, AGR2 overexpression leads to pro tumourigenic effects, including increased proliferation and survival.