Calpains and mammary gland: The project

  BACKGROUND

 

1.1 Calpains as pleiotropic processing enzymes.

Calpains (CAPN) are Ca²⁺-dependent Cys-proteases involved in a wide variety of physiological and pathological processes, including post-lactation mammary gland involution and breast cancer, respectively.

Among the 15 different genes coding for calpains, CAPN1 and CAPN2 (also known as μ-calpain and m-calpain, respectively) are the most ubiquitously expressed isoforms in mammalian tissues. Both calpains form heterodimers composed of a unique catalytic subunit (80KDa) and a common regulatory subunit (30–29KDa) named CAPN4 [1].

Initially considered as mere degradative enzymes, the processing role of CAPNs has gained special relevance today. CAPNs, in a strictly regulated manner, and in response to specific stimuli, will process proteins, so that their end-products can acquire new functions, distribution or forms of regulation [2]. In fact, CAPNs have been compared to a post-translational splicing system. Accordingly, CAPN functions will depend on their substrates and end-products. In addition, although long ago considered as redundant enzymes, the isoform-specific role of CAPNs is currently accepted [1-6]. Accordingly, the identification of isoform-specific substrates of CAPNs as well as their end-products seems to be a key step to define their functions.

 

To date more than 200 proteins have been reported to be recognized and cleaved by CAPNs in vitro [2], which explains the large number of physiological or pathological processes they modulate [1]. However, substrate-recognition by CAPNs is a process poorly understood. In contrast to other proteolytic systems, CAPN-mediated cleavage of proteins does not require a prior post-translational modification, such as ubiquitination of proteins to target them for proteasome degradation. Furthermore, although a target sequence for CAPNs has been described, they also recognize secondary structures adjacent to the cleavage-site. These data explain why the isoform-specific role of CAPN1 and 2 has not been accurately elucidated. Indeed, both calpains recognize the same substrates in vitro and yet they have different functions in vivo. The determinants for substrate recognition must be more complex than thought at first. Actally, CAPNs functions are reported to be highly dependent on the cell type, the type of stimulus and the biological process in which they participate [4-8].

 

Given their pleiotropic effects, CAPNs could be considered as important regulatory nodes: according to the variety of proteins processed by them, their effects would be expected to be exponentially amplified by their end-products. Furthermore, it is well known that different molecular events concurrently converge in the same biological process. For example, during embryogenesis, processes such as cell division, adhesion, differentiation, and cell death are simultaneously produced and coordinated by regulatory nodes. The role of CAPNs during embryonic development has been explored in knockout mice. While the CAPN1-deficient mice are viable and fertile [9], CAPN2-knockout mice resulted embryonically lethal [10]. These data not only demonstrate the isoform-specific role of CAPNs, but most importantly, it suggests that the role of CAPN2 as a regulatory node could be extended to the coordination among the different molecular processes taking place within the same tissue. In fact, although the physiological role of CAPN2 is not completely understood, it is known to be involved in variety of processes, going from the modulation of cell survival and cell growth, to cell proliferation, angiogenesis or cell migration [1, 4-8].

 

1.2.Physiological role of Calpains in post-lactating mammary gland involution

The pregnancy/lactation cycle of mammary gland is a good example of sequential and overlapping molecular processes taking place in a single tissue. Processes such as the regulation of cell fate, cell and tissue polarity, morphogenesis, cell proliferation, cell death, cell differentiation or tissue remodeling [11] should be finely coordinated in each pregnancy/lactation cycle. Therefore, the pregnancy/lactation cycle of murine mammary gland is an excellent model to study CAPN2 as a node for the regulation of different biological processes tightly coordinated in different cell types within the same tissue [12]. In this sense, it is important to highlight that most reports about CAPNs functions use cultured cells. However, cultured cells do not resemble the polarized acinar structure and, tissue organization is not an issue to be overlooked when studying the physiological role of a protein known to have cell type and context-dependent functions.

 

Mammary gland tissue is composed by epithelia cells (luminal, alveolar and myoepithelial) macrophages and mesenchymal cells, including adipocytes, fibroblasts and endothelial cells. During puberty, the rudimental mammary gland formed during the embryonic stage is expanded to invade the subjacent mesenchymal tissue and a tubular branched system sustained by fat will be formed. In each pregnancy, the mammary gland will cyclically go through a set of cellular and molecular changes:

 

• During the pregnancy/lactation cycle hormonal changes induce the proliferation, expansion and branching of alveolar cells which, upon maturation and differentiation, will form the milk-secreting acini [11-13]. Although CAPN1 is the most abundant isoform, both CAPNs are expressed at the peak of lactation [4]. However, while CAPN1 localizes at the apical region of epithelial cells, CAPN2 is localized at cell-cell adhesion regions of basal cells and within some nuclei [6]. Differences in distribution of both CAPNs during lactation also suggest functional differences not yet explored.
• Upon cessation of lactation, during mammary gland involution, milk stasis and drop of lactogenic hormones promote important changes leading to the inhibition of milk production, induction of secretory cell-death and stimulation of a pro-inflammatory environment. A controlled flow of macrophages and other immune cells to the mammary gland would clear dead milk-secreting cells. After 48h involution, during the second and irreversible phase of involution, extracellular matrix is disorganized, blood vessels remodeled and adipocytes re-differentiated to regenerate mesenchymal tissue and return the mammary gland to a pre-pregnant-like state [13].

It was classically thought that at this phase, proteases such as MMPs (matrix metalloproteinases), cathepsins and CAPNs would completely remodel the tissue. However, the role of CAPNs goes further than their role as degradative enzymes. The expression and enzymatic activity of both CAPN-1 and -2 are induced during the first 48h of involution and last throughout the second stage of involution [4-6]. Their cellular and subcellular distribution and consequently, their role in the different stages of mammary gland involution might be complementary, but considerably different :

On the whole, these data suggest that the functions of CAPNs are specific for each isoform and that they depend on the stimulus, cell type and subcellular distribution, but above all, they could play an important role as a regulatory node. The existence of common regulatory nodes for various processes is not a new idea. In this sense, the transcription factor NFkB has been postulated as one of the key regulatory nodes for the pregnancy/ lactation cycle of mammary gland [14]. Interestingly, we have previously reported the modulation of CAPN1 and -2 expression by this transcription factor during early involution of mammary gland after lactation [4].

 

1.3 Calpains from physiological mammary gland to breast cancer

Many of the proteins considered as regulatory nodes have been identified as persistently activated oncogenes or transcription factors during neoplasia transformation. Such is the case for the transcription factor NF-κB, which plays an important role in inflammation, cell survival, proliferation and oncogenesis. It is interesting to highlight the fact that the mammary gland during post-lactation involution shows many similarities with the gene expression pattern and the activation of signaling pathways described in cancer [12, 14, 15]. In this sense, it has been suggested that the pro-inflammatory environment of mammary gland involution could promote tumor progression [14-16].

CAPNs dysregulation in cancer have been extensively reported [17]. However, data are controversial and a prognostic value for CAPNs in breast cancer could not be definitely stablished [17-19]. High levels of CAPN expression have been associated with breast cancer [18]. A correlation between CAPN1 expression and poor prognosis or even resistance to trastuzumab treatment in breast cancer has been described, but no correlation has been found with the expression of calpastatin, the endogenous inhibitor of both calpains, or with CAPN2 levels [18,19]. It has been argued that discrepancies among reports are most probably caused by the type of analysis performed in these clinical studies, and the different classification of breast tumor subtypes. Importantly, most of these reports only study levels of CAPN expression, and no reports of enzymatic activity or subcellular distribution of CAPNs have been reported [1]. In this sense, combining analysis of mammary gland involution and cultured breast cancer cells, we obtained evidence supporting the notion that the subcellular distribution of isoform-specific CAPNs can switch upon neoplastic transformation [6]. CAPN2 identified as the membrane-associated isoform involved in cell detachment of epithelial cells during mammary gland involution, was restricted to the nuclear compartment of breast cancer cells and, CAPN1 was found to be the main isoform mediating migration of these cells independently of the cancer subtype [6].

CAPN2 accumulates in the nucleus and nucleolus of human breast cancer cells [8]. Since the same subcellular distribution was also found in human colorectal cell lines [7], nuclear accumulation of CAPN2 does not seem to be cancer-type specific. Several studies have shown that while in differentiated quiescent cells CAPN2 is restricted to the cytosol [20], in proliferating cells, CAPN2 is mainly localized in the nucleus [20-22], Moreover, nuclear localization of CAPN2 has been associated to high expression levels and active mitosis in embryonic stem cells as well as in 8-cell embryos [21].

The role of CAPN2 in the nucleus of proliferating cells is not yet entirely clear. Our studies show that nucleolar CAPN2 is involved in ribosomal biogenesis by the regulation of rDNA transcription [7] and in other processes not related to ribosomal biogenesis [8]. Indeed, CAPN2 depletion in breast cancer cells resulted in aberrant mitosis and cell multinucleation [8] suggesting a role in cytoskeleton remodeling and mitosis, maintenance of nuclear structure, the movement of chromosomes and the modulation of transcription frequently altered in cancer cells. However, these undoubtedly important data in breast cancer cell lines do not reflect the context of mammary gland in vivo.

As mentioned above, the pro-inflammatory microenvironment of mamma gland involution after lactation is known to favor tumor progression. A paradoxical concept, since it was historically understood that the pregnancy/lactation cycle

protected against breast cancer development. However, a prevalence study has recently published that breast cancer risk increases during the 5 years following parturition [23]. This increase will be thereafter reversed or even decreased below that found in nulliparous women. The same study reports that pregnancy-associated increase in breast cancer risk becomes more pronounced with increasing age at first pregnancy. The pregnancy-associated raise in breast cancer risk is not reversed and counts for the overall increase of breast cancer risk in women older than 35 years at their first full-term pregnancy (Figure 2) [12].

The breast cancer risk associated to age at first pregnancy results specially alarming in a model of society in which the age to have offspring is progressively postponed. According to the latest data published by Eurostat (2019), the average age of European women for their first child is over 30 years old, with Spain (average age 32.3 years) in the third position of European countries with older ages for first pregnancy. This fact, which is alarming enough by itself, is even worse when the progression in the last 10-year is analyzed. This analysis suggests that the age to have offspring will follow the same or higher trend in the coming years. Consequently, the study of the underlying and poorly understood mechanisms explaining these epidemiological data are extremely relevant. However, murine models used to study the parity-associated protection of breast cancer do not take into consideration the age of mice at first pregnancy.

In addition to early pregnancy, multiple pregnancies and breastfeeding also decrease breast cancer risk [23]. However, it was recently reported that parity-induced reduction of breast cancer risk accounts only for the ER-positive subtype. For the triple negative TNBC subtype, parity may increase the risk of breast cancer [24]. This risk seems to be reduced by breastfeeding. Data from AMBER consortium study showed increased risk of TNBC by increasing parity in women who never breastfeed [25].

Nevertheless, duration of breastfeeding is another important factor to be considered. A large collaborative study with data from 32 different countries have reported a 28% reduction of breast cancer risk in parous women who breastfed for a period ³12 months and only a 14% reduction in women breastfeeding for a shorter time compared to parous women who never breastfed [26]. In agreement with this, a recent study shows that forced weaning induces morphological changes in the murine mammary gland after short lactation which were not evident in the long-lactation mice [27]. Mammary gland from the short-lactation group showed denser stroma, increased type collagen deposition, higher inflammatory response and increased cell proliferation rate than the long-lactation group. Furthermore, mammary gland from the short-lactation group exhibited ductal hyperplasia and squamous metaplasia at 4 months after parturition, both preneoplastic conditions for breast cancer and accordingly a breast cancer risk factor.

All these data together do not allow promising expectations for the future. Women postpone their reproductive life and reduce the period of lactation for work reasons.  An exhaustive study of those molecular mechanisms underlying the pregnancy/lactation cycle of mammary gland should take into account the age at first pregnancy and the length of lactation: an experimental model better representing the current reproductive life of women and child-feeding, which has never been studied. Accordingly, the role of CAPN2 or any other regulatory node described for mammary gland involution after lactation have been studied (by our group or others) only in experimental models of 10-weeks old mice and short-lactation. Female mice will begin their reproductive life at 6 weeks old, so 10-weeks old mice could be considered the equivalent to 20-25-year old human female.

Breast cancer risk factors associated to the age at pregnancy and duration of lactation have been dismissed or underseen in those previous murine models. Since CAPN2 activity, subcellular distribution and functions is highly dependent on the tissue context, we believe that the age at first pregnancy together with the duration of lactation could have an impact on CAPN2 dysregulation. Furthermore, most reports on the pregnancy/lactation cycle of mammary gland study changes at 7-10 days after forced involution, missing possible changes ongoing in the mammary gland thereafter (such as those exhibited by parous women).

 

2. REFERENCES

 

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3. Our aim