Am J Res 2018;8(11):2150-2164 www.ajcr.us /ISSN:2156-6976/ajcr0085184

Review Article Prolactin-induced (PIP)-characterization and role in breast cancer progression

Anna Urbaniak1,2, Karolina Jablonska2, Marzenna Podhorska-Okolow2, Maciej Ugorski1,4, Piotr Dziegiel2,3

1Laboratory of Glycobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; 2Department of Human Morphology, Division of Histology and Embryology, Wroclaw Medical University, Wroclaw, Poland; 3Department of Physiotherapy, University School of Physical Education, Wro- claw, Poland; 4Department of Biochemistry and Molecular Biology, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland Received September 7, 2018; Accepted September 17, 2018; Epub October 1, 2018; Published October 15, 2018

Abstract: Prolactin-induced protein (PIP) is a small secreted glycoprotein carrying several N-linked carbohydrate chains. The expression of PIP is generally restricted to cells with apocrine properties. It was found in apocrine glands of the axilla, vulva, eyelid, ear canal, and seminal vesicle. Being a secretory protein, PIP is present in seminal plasma, saliva, lacrimal fluid, tears, sweat gland secretion. Little is known about the biological role of PIP. It binds to numerous , however, in most cases the biological role of such interactions is poorly understood. A notable exception is its binding to CD4 receptors present on the surface of T lymphocytes, macrophages, and spermatozoa. The available data suggest that PIP can have immunomodulatory functions and plays an important role in cell- mediate adoptive immunity. PIP binds to bacteria from several genera, which suggests that this glycoprotein may participate also in innate immunity and protection of hosts against microbial infections. Increased levels of PIP were found in several types of human cancer (prostate, sweat and salivary gland ). It is especially common in breast cancer, however, data on the expression of PIP in normal and cancerous breast cancer tissues are to some degree conflicting. In early studies, it was shown that PIP is absent or its expression is very low in normal breast epithelium, whereas in breast cancers PIP is frequently expressed and present in large amounts. On the other hand, later study showed that expression of PIP is lower in advanced apocrine carcinomas and invasive carcinomas than in, respectively, in situ carcinomas and adjacent normal tissue. The most recent study revealed that PIP ex- pression decreased gradually along with higher stage and grade of breast cancer. In agreement with these data, it was shown that that low levels or the lack of PIP expression are associated with a worse response of breast cancer cells to chemotherapy. It was proposed that PIP plays important role in the development and progression of breast cancer. However, its role in these processes is both unclear and controversial. In this review, the role of PIP in both physiological processes and carcinogenesis is discussed.

Keywords: PIP, GCDFP-15, gp-17, prolactin-induced protein, gross cystic disease fluid protein 15, breast cancer

Introduction ultimately lead to naming it prolactin-induced protein (PIP). Subsequent study confirmed that At the end of the 20th century, Haagensen it is the same protein as GCDFP-15 [4, 5]. Sh- and Mazoujian described Gross Cystic Disea- ortly after these reports, independent research se Fluid Protein 15 (GCDFP-15) as one of the showed that PIP/GCDFP-15 under the name gly- proteins present in the cystic fluid from mas- coprotein-17 (gp-17) and seminal actin-binding topathy [1, 2]. A few years later, Shiu and Iwa- protein (SABP) was present in the glandular epi- siow identified acidic protein that was present thelium of seminal vesicles and as extra parotid in the culture medium from breast cancer T47D glycoprotein (EP-GP) was found in submandibu- cells as two glycoforms of molecular masses lar and sublingual glands [6-8]. Presently, the 16 kDa and 14 kDa [3]. They found that the commonly accepted name for all these proteins level of this protein was significantly increas- in human and mouse genomic nomenclature is ed following prolactin (hPRL) induction, which PIP. Urbaniak et al.: PIP in breast cancer

Expression of PIP under physiological and in situ carcinomas [29]. Similarly, significantly pathological conditions lower levels of PIP were found in invasive bre- ast tissue then in adjacent normal tissue [30]. The expression of PIP is generally restricted to In agreement with these data, the most re- cells with apocrine properties [9, 10]. Using cent study revealed that PIP immunohistochemistry, it was found in apo- decreased gradually along with higher stage crine glands of the axilla, vulva, eyelid, ear and grade of breast cancer [31]. The authors canal, and seminal vesicle [11]. However, PIP showed that PIP mRNA and protein expression was also detected in serous cells of salivary in normal breast tissue were significantly high- glands, submucosal glands of the bronchi, er than in breast cancer tissues. Significant and accessory lacrimal glands [9, 11]. These downregulation of PIP was also observed in tissues do not represent typical apocrine epi- early stages of breast tumor progression. thelia, but phylogenetically have common prop- erties with apocrine glands. Being a secretory With the introduction of new molecular clas- protein, PIP is present in seminal plasma, sali- sification based on gene expression profiling, va, lacrimal fluid, tears, sweat gland secretion, the following molecular subtypes of breast amniotic fluid, and the blood of pregnant wo- cancer were discriminated: basal-like, HER2- men [2, 12, 13]. enriched, luminal A, luminal B and normal-like [32-34]. According to this nomenclature, the PIP is generally expressed by normal breast highest amounts of PIP mRNA were found in tissues, but is highly present in metaplastic/ the luminal A subtype, then in HER2-enriched hyperplastic apocrine epithelium of breast cyst and normal-like subtypes, and the lowest ex- and breast cyst fluid [1, 2, 9, 14, 15]. Recently, pression was observed in basal-like subtype it was shown that in keratoconus disease, [35, 36]. In another study based on analysis the expression of PIP is highly diminished in of global gene expression, which identified mo- keratoconus cells and tears, therefore it was lecular apocrine breast cancer subtype [37], it proposed that PIP can be a potential biomar- was found that such tumors are characterized ker for this disease [16]. The fact that signifi- by increased amounts of PIP [38]. This expres- cantly decreased expression of PIP was found sion strongly correlated with the presence of in tape-stripped stratum corneum and sweat AR [36, 39, 40]. Since in many studies, PIP samples from atopic dermatitis patients, sug- expression correlated with low grade breast gests the possibility that PIP may also be a cancers, it was proposed that high expression marker for atopic dermatitis associated with of this marker is a predictor of good prognosis decreased eccrine sweating [17]. [25, 39, 41, 42]. From clinical point of view, is also important that cases with high PIP ex- PIP in breast cancer pression were characterized by longer disease- free survival [43-45] and overall survival [45]. In early studies, it was shown that PIP is ab- In our published study, we showed that a high sent in normal breast epithelium or its ex- level of PIP expression is positively correlated pression is very low and/or difficult to detect, with the response of breast cancer patients whereas in breast cancers PIP is frequently to standard adjuvant chemotherapy (doxorubi- expressed and present in large amounts [18- cin + cyclophosphamide) [46]. The data ob- 21]. Several early studies revealed that PIP is tained indicates that low levels or the lack of especially present in breast carcinomas with PIP expression are associated with a worse apocrine features [11, 22-24]. Its expression response of breast cancer cells to chemothera- was also confirmed on mRNA and the protein py. Expression DNA microarray studies show level in axillary node metastases [18-20, 24- that the expression of the PIP gene is signifi- 27]. However, others have shown that PIP cantly lower in cases of invasive ductal carci- mRNA was more frequently present in unin- noma (IDC), which are poorly responsive to volved breast tissue then in breast carcinoma, standard adjuvant chemotherapy [46] (Figure and metastatic breast carcinoma [28]. Also 1). Additionally, we showed that the levels of later studies showed that expression of PIP PIP protein and mRNA decreases along with decreases in advanced apocrine carcinomas tumor malignancy grade (G), and that PIP ex- and was significantly lower in infiltrating carci- pression is the lowest in triple negative (ER-, nomas, especially node-positive tumors, than PR-, HER2-) cases with poor prognosis.

2151 Am J Cancer Res 2018;8(11):2150-2164 Urbaniak et al.: PIP in breast cancer

Figure 1. Images of immunohistochemical reactions showing expression pattern of PIP in IDC cells. PIP protein was localized in cytoplasm of tumor cells. (A) Low PIP expression, (B) Medium and (C) High intensity of PIP expression. Magnification ×200.

plasma from patients with pri- mary and metastatic breast Figure 2. A. The three- cancer [1, 19, 55]. dimensional structure of PIP. PIP is composed of PIP protein seven β-sheets. The po- tential N-glycosylation site Human PIP is synthesized as is at position Asn77. The 146 amino acid pre-protein tertiary structure of this protein contains two di- [4]. Following cleavage of 28 sulphide bridges formed amino acid signal peptide, PIP between Cys37 and Cys63 becomes an 118 amino acid and Cys61 and Cys95 secretory polypeptide of the [105, 106]. B. The amino theoretical molecular mass- acid sequence of PIP and 13,506 kDa. SDS-PAGE ex- localization of fibronectin (FN)- and CD4-binding do- periments showed PIP appar- mains [60-62]. ent molecular mass to be 14-20 kDa, depending on the origin of the protein [1, 12, 56, 57]. The difference be- tween theoretically and ex- perimentally determined mo- lecular masses is a result of N-glycosylation, and differe- nces in apparent molecular masses of PIPs isolated from various sources are most As PIP was not found in gastrointestinal can- probably caused by variations in the structure cers, bronchopulmonary structures, and geni- of N-glycans (see below). Microheterogeneity tourinary cancers, this specific marker of breast linked to N-glycosylation is also reflected by cancer and its metastases can be used to dis- differences in isoelectric points ranging from tinguish distant breast metastases from other pH 4.8 to pH 5.4 [13, 57]. At its N-terminus, primary and secondary tumors [21, 41, 47-53]. PIP has a cyclic glutamine derivative-pyrogluta- Furthermore, PIP may be a potential marker mine [12]. for breast micrometastases to auxilliary lymph nodes [25]. However, it should be remember- In 2008, Hassan and co-workers determined ed that PIP was also found in cancers of pros- the crystal structure of PIP in complex with ZAG tate, sweat and salivary glands [20, 54]. Highly protein (zinc-alpha-2-glycoprotein) [58] (Figure increased levels of PIP, in comparison to nor- 2A). They showed that PIP is composed of mal subjects, were observed in the peripheral seven parallel β-sheets and seven β-turns.

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Figure 3. Schematic representation of the PIP gene [64].

Characteristic for PIP is the lack of α-helix- (gp17/SABP), CD4-binding domain was identi- type structures. As a result of the highly hydro- fied as two fragments encompassing the 1-35 phobic character of amino acid residues, β-sh- N-terminal amino acids and 78-105 amino eet motifs are organized as pairs in the form of acids, and fibronectin-binding domain as two hollow, sandwich-type structures. The spatial peptides encompassing 109-118 C-terminal structure of PIP resembles the 7th fibronectin amino acids and 42-57 amino acids [60-62]. type III domain, although this domain has only 13% identity with PIP sequence. PIP tertiary PIP gene and regulation of its expression structure analysis showed the presence of two disulphide bridges formed between cysteine The PIP gene is localized on the long arm of residues at the positions 37 and 63 and 61 7 at locus q34 and includes 7000 and 95 [12, 58]. PIP may form dimers or ag- base pairs (bp) [5, 63]. It consists of four exons gregates of 4-12 monomers [57, 59]. and three introns [64] (Figure 3). In the pro- moter region a classical TATA box and a CAAT In PIPs isolated from human saliva and breast box were identified. The methylation revealed cystic fluid (PIP/GCDFP-15) the carbohydrate that methylation is probably necessary, but content was found to be, respectively, 13.2%, not sufficient for expression of the PIP gene and 8.5% [56, 57]. In the case of the latter, the [64]. Interestingly, genetic alterations within single N-glycan linked to Asn77 represents a are commonly found in human complex-type structure with an unusually high breast cancer [65, 66]. In line with this, Ciullo content of fucose [56]. However, PIP/SABP et al. found that the breakage of chromosome from seminal plasma was characterized by a 7 in the FRA7I area caused reversed palindrom- much higher carbohydrate content (22%). The ic PIP gene duplication in T47D cells, which single N-glycosidic carbohydrate chain proba- probably is the reason for increased, constitu- bly represents complex-type structure [12]. A tive PIP expression in these cells [67]. Also it more recent study showed that N-glycans from was shown that the intragenic region of the PIP/SABP are monosialylated diantennary stru- PIP gene is involved in the formation of small ctures with various fucosylation patterns, and polydispersed circular DNA, and such DNA mol- are different from N-glycans linked to PIP/GC- ecules may serve to enhance genetic instability DFP-15, identified as triantennary structures [68]. [60]. According to those authors, differences in carbohydrate structures suggest that higher The nucleotide sequence of PIP mRNA was degree of sialylation in pathological form of determined for the first time by sequencing PIP may better protect this glycoprotein from cDNA isolated from expression library which proteolytic degradation and affects the upta- was constructed from mRNA from breast can- ke and processing of sialylated glycoproteins cer T47D cells [4]. It was found that PIP mRNA by immune cells. represented a single transcript of about 591 nucleotides long (NCBI refference seq. NM_ In addition to the actin-binding motif, which 002652.2 16.04.2018). was found by in silico analysis [6], PIP show- ed the presence of CD4- and fibronectin-bind- There are few reports on the evolution of the ing domains, which were found experimentally PIP gene [69]. A comparison of nucleotide se- (Figure 2B). In PIP from human seminal plasma quences in primates [chimpanzees (Pan troglo-

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bridges [70]. A phylogenetic tree indicates similarity be- tween the PIP gene and ge- nes coding such proteins as A2M (alfa-2-makroglobu- lin), PZP (pregnancy-zone-pro- tein), A2ML1 (alpha-2-macro- globulin like 1) and OVOS2 (ovostatin 2) [62].

The expression of the PIP gene on the levels of mRNA and protein is increased by androgens, progesterone, glu- corticosteroids together with prolactin or growth hormone [3, 9, 19, 71-74], and cyto- kines such as IL-1α, IL-4 and IL-13 [75, 76] and decreased by 17β-estradiol and IL-6 [74, 77, 78].

There is evidence that PIP expression is primarily regu- lated at the transcriptional level. Originally, it was shown that PIP gene expression in ZR-75 cells was highly incre- ased by the simultaneous action of 5α-dihydrotestos- Figure 4. A. Regulation of PIP expression. 1. Synergistic action of prolactin terone and prolactin, which and 5α-dihydrotestosterone leads to an increased PIP expression by activa- bind, respectively to the an- tion of STAT5 and AR transcription factors that bind to the promotor region of drogen (AR) and prolactin the PIP gene. 2. Synergistic action of AR activated by 5α-dihydrotestosterone (PRLR) receptors [79]. Pro- and Runx2 that bind to the promotor region of the PIP gene leads to in- lactin binding induces phos- creased PIP expression [80, 81]. AR-androgen receptor, ARE-androgen re- sponse element, DHT-dihydrotestosterone, ErbB2-Her2, human epidermal phorylation of STAT5A or/and growth factor receptor 2, Jak-Janus-activated kinases, PIP-prolactin-induced STAT5B, which after dimeriza- protein, PRLR-prolactin receptor, Runx2-Runt-related transcription factor 2, tion are translocated from STAT5-signal transducer. B. At the promoter site of PIP gene, regions I, II, III submembranous localization and IV are located -0.9 kb, -2.4 kb, -9.4 kb, and -11 kb from the transcription initiation site [80]. into the nucleus. There they bind to the STAT5-responsive element located on the PIP dytes), gorillas (Gorilla gorilla), orangutans (Po- gene promoter and cooperate with activated ngo pygmaeus), gibbons (Hylobates agilis)] and AR bound to androgen responsive elements of humans suggests that the PIP gene could have PIP promoter to increase the transcription of evolved as a result of positive selection caused the PIP gene (Figure 4A1). A similar regulatory by interaction between PIP protein and patho- mechanism, which involves cooperation of AR gens [62]. It was also found that in hominoids and Runx2 transcription factor was described amino acid changes accumulated in the second by Baniwal et al. in human breast cancer T47D fibronectin-binding domain (FN2), in contrast to cells and prostate cancer C4-2B cells [80]. the conserved first CD4-binding domain (CD4- According to them, following activation by 5α- 1) (see section 4). Particularly evolutionary con- dihydrotestosterone, AR and Runx2 bind to- served among mammals are regions encoding gether to enhancer element of PIP promoter four cysteines that form two pairs of disulphide and by physical interaction act in a synergistic

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vates Akt/PkB and ERK sig- naling pathways, while ErbB2 binding activates MAPK/ERK signaling. Both of these both signaling pathways, by phos- phorylation of the RSK and MSK families of kinases, by phosphorylation activate the CREB1 transcription factor, which binds to the PIP gene promoter and increases its transcription. In ER-negative breast cancer cells, PIP ex- pression is also regulated by positive AR, ERK positive fe- edback loop, since CREB1 also activates transcription of the AR gene. The resulting AR protein increases expres- sion of ErbB2, which in turn activates the ERK, CREB1 axis.

Figure 5. Regulation of PIP expression. In ER-negative breast cancer, the ex- In summary, all the available pression of PIP is auto-regulated by the positive feedback loop between PIP and ERK, Akt/PkB signaling [81, 107, 108]. AR-androgen receptor, CREB1- data shows that positive re- cAMP responsive element binding protein 1, DHT-dihydrotestosterone, gulation of PIP expression in ErbB2-Her2, human epidermal growth factor receptor 2, ERK1/2-extracel- breast cancer cells on the lular signal-regulated kinases 1 and 2, ILK1-integrin-linked kinase 1, Jak- level of transcription is de- Janus-activated kinases, MSK-mitogen- and stress-activated protein kinase, pendent on the cooperation PIP-prolactin-induced protein, PKB/AKT-protein kinase B/serine/threonine of AR with different transcrip- kinase, PRLR-prolactin receptor, RSK-ribosomal S6 kinase, Runx2-runt-relat- ed transcription factor 2, STAT5-signal transducer. tion factors, such as STAT5, Runx2, and CREB1. manner to highly increase the expression of the Biological functions of PIP PIP gene. Runx2 and AR are recruited to four regions in the promoter sequence of PIP gene PIP has an aspartyl protease activity [82]. This (Figure 4A2). Regions I, II, III and IV (R-I-IV) are function results from the presence of asparta- located, respectively, -0.9 kb, -2.4 kb, -9.4 kb, te residue at position 22 (Asp22), which shows and -11 kb from the transcription initiation site. homology to aspartate residue 32 in other Regions I and II include consensus sequences known aspartyl proteases, such as cathepsin for Runx2, whereas regions III and IV contain D, pepsin or renin. As fibronectin is one of the consensus sequences for Runx2 and AR (Fi- substrates of PIP, it is believed that PIP par- gure 4B). In turn, PIP positively regulates an- ticipates in extracellular matrix degradation, drogen signaling, facilitating translocation of and therefore is engaged in breast cancer pro- AR to the nucleus and stimulation of androgen- gression. In addition to fibronectin, PIP binds dependent . numerous proteins including: actin, fibrinogen, β-tubulin, serum albumin and hydroxyapatite In ER-negative breast cancer, the expression of (a major component of tooth enamel), zinc α2- PIP is auto-regulated by the positive feedback glycoprotein and Fc fragment of IgGs [8, 12, loop between PIP and ERK, Akt/PkB signaling 36, 57, 83-85]. However, in most cases the (Figure 5) [81]. In human molecular apocrine biological role of such interactions is poorly breast cells, the secreted PIP, by its proteolytic understood. activity degrades fibronectin to peptides (see section 5), which activates β1-integrins to in- PIP/EP-GP from human saliva binds to bacteria teract with integrin-linked kinase 1 (ILK1) and from the genera Gemella, Streptococcus and ErbB2 (Her2-neu) molecules. ILK1 binding acti- Staphylococcus, and causes their aggregation

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[8, 86, 87]. Similar properties were shown by cytic aggregations within the prostate lobes, murine salivary PIP, which bound to bacteria and an enlargement of thymic medulla, howev- from the genus Streptococcus [88]. Based on er, their development was normal and animals these results, it was suggested that PIP is a were fertile [94]. knock-out mice revealed the part of the oral defense mechanism against presence of many differentially expressed ge- bacterial pathogens. The idea that PIP partici- nes related to cell death and survival, inflam- pates in innate immunity and protection of host mation, immune response and cancer in com- against microbial infections is supported by the parison to parental mice. This further supports fact that it is present in significant amounts in the involvement of PIP in immunological pro- mucosal-type tissues, bronchial submucosal cesses. The loss of PIP expression in mice glands, apocrine glands of the skin, saliva, and leads to a significant decrease in the CD4 posi- lacrimal fluid, all of which represent ports of tive T cell population in spleen, and has a nega- pathogen entry. tive impact on their differentiation to CD4 posi- tive Th1-cells, resulting in a highly decreased Several studies revealed that PIP isolated from production of IFNγ [95]. As the result of low different sources such as human seminal fluid numbers of CD4 positive Th1-cells, such mice (gp17/SABP) and breast cyst fluid (GCDF-15) are highly susceptible to Leishmania major in- binds to CD4 receptors present on the surface fection. Taking into account that differentiation of T lymphocytes, macrophages, and spermato- of Th1 cells and the outcome of infection zoa [7, 59, 60, 89]. However, using surface depend on the production of IL-12 by antigen plasmon resonance (SPR), it was found that presenting cells (APC), the authors found that PIP/GCDF-15 derived from pathological sourc- bone marrow derived macrophages from PIP- es (breast cyst fluid) binds to CD4 with lower knock-out produce significantly lower amounts affinity than physiological form of PIP/gp17/ of IL-6, IL-12p40 and TNF than wild-type ani- SABP from human seminal plasma [60]. These data suggest that depending on the tissue and mals after in vitro stimulation with LPS and physiological v. pathological conditions, PIP polyI: C. These findings suggest that PIP indi- may interact differently with other molecules rectly affect maturation of naive CD4 positive and therefore can have distinct functions. CD4 T cells to Th1-cells by decreasing production plays a key role in immune response, acting as of cytokines by antigen presenting cells. The TCR co-receptor essential for recognizing pep- increased sensitivity of PIP-knock-out mice to tides presented by major histocompatibility L. major infection is also associated with highly complex II (MHC II) on the antigen presenting decreased production of NO by bone marrow- cells [90]. This molecule is also involved in derived macrophages after LPS and IFNγ stimu- T cell activation by interaction with the pro- lation. It was found recently that decreased tein-tyrosine kinase p56lck [91]. CD4 is a recep- production of pro-inflammatory cytokines by tor for human immunodeficiency virus (HIV-1) macrophages from PIP-negative mice is associ- gp120 envelope glycoprotein and it has been ated with decreased phosphorylation of mito- suggested that such interaction can increase gen-activated protein kinase (MAPK) and signal HIV-induced T cell apoptosis [92]. The binding transducer of activation of transcription (STAT) of PIP to CD4 inhibited its interaction with proteins. In such mice, the expression of sup- gp120 and syncytium formation by cell express- pressors of cytokine signaling (SOCS) 1 and 3 ing gp120 and CD4, which strongly suggests proteins was higher than wild type mice [96]. All that PIP may be involved in pathogenesis of these data further support the idea that PIP HIV-1 infection [92]. In fact, further study re- plays an important role in cell-mediated immu- vealed that PIP strongly inhibits T cell apoptosis nity. Independently, it was also shown that the induced by sequential gp120/CD4 and TCR/ level of PIP expression increases following stim- CD3 activation [93]. It was also shown that the ulation with IL-4 and IL-13, which play a key role binding of gp17 significantly increased the ex- in the regulation of immune cells activity [75]. pression of anti-apoptotic Bcl-2 protein. These data suggest that PIP can have immunomodu- PIP protein, present in high amounts in human latory function in some virus infections and seminal plasma, is an IgG-reacting protein, generally modulate adaptive immune response. which binds to the Fc fragment of antisperm antibodies (ASAs) that recognize seminal pro- PIP gene knockout mice showed enlarged lym- teins and can cause infertility in men [84]. ph nodes around the parotid glands, lympho- But the total levels of PIP as well as levels of

2156 Am J Cancer Res 2018;8(11):2150-2164 Urbaniak et al.: PIP in breast cancer different PIP isoforms in seminal plasma did tion, which correlated with a reduction in ex- not correlated with fertility status. This, how- pression levels of cyclin D1 and cyclin E1 ge- ever does not exclude its immunoregulatory nes, and decreased phosphorylation of ERK. A functions. second group of cell lines, comprised of MFM- 223, SK-BR3, HCC-1954, HCC-202 and BT-474 The role of PIP in breast cancer progression cells after PIP gene knockdown, was character- ized by a significant increase in the G2/M phase The biological functions of PIP were primarily and the percentage of aneuploidy, which was studied using established in vitro breast can- associated with decreased expression of cyclin cer cell lines. Early studies performed on tu- D1 and cyclin B1 genes, a reduction in total and mor cells of different origin revealed that only phospho-Cdc2 protein levels, and a significant breast cancer cells respond to exogenous PIP decreased in FOXM1, TTK, BUB1, and CDC20 by increased proliferation [97]. The important genes expression. In summary, these observa- role of PIP in proliferation of breast cancer cells tions suggest that in breast cancer cells PIP is was further confirmed by knockdown of the involved in the progression of cell cycle, espe- PIP gene in T47D and MDA-MB-453 cells [80, cially during the mitotic phase primarily as a 81]. The inhibition of PIP expression highly de- regulator of the transcription of key cell cycle creased the proliferative potential of such cells. genes. Proteomic studies performed on the Interestingly, with T47D cells, proliferation stim- same cells revealed that PIP interacts with β- ulated by serum growth factors or dihydrotes- tubulin and facilitates tubulin polymerization, tosterone was fully dependent on the presence which can further explain its role in cell prolif- of PIP [80]. When more breast cancer cell lines eration, since microtubules are involved in mi- with PIP knockdown were studied, it was found totic transition, spindle assembly and cytokine- that generally proliferation of cell lines repre- sis. PIP also interacts with Arp2/3 protein, whi- senting luminal A, luminal B and apocrine sub- ch participates in actin polymerization and fa- types of breast cancer is dependent on the cilitates talin binding to integrins, which can presence of PIP [36]. explain why PIP is required for inside-out ac- tivation and signaling effects of integrin-β1. In Studies on the molecular mechanisms conne- contrast to the results of Cassoni et al. (1995), cting PIP expression with increased prolifera- the studies on breast cancer cells with silenc- tive potential of breast cancer cells revealed ed PIP suggest that stimulated effects of PIP that PIP knockdown was associated with de- on their proliferative potential are mediated by creased phosphorylation of focal adhesion ki- intracellular PIP interacting with cytosol pro- nase (FAK), ephrin B3 (EphB3), tyrosine kinase teins, not an exogenous form of PIP. However, of the Src family (FYN), and hemopoietic cell this suggestion should be treated with caution kinase (HCK) [35]. The absence of PIP also because electron microscopy showed that PIP made the activation of serine/threonine kinas- is present in secretory vesicles [98], and there- es AKT, ERK1/2 and JNK1 by serum factors fore its presence in cytoplasm is rather unlike- impossible. Such changes in intracellular sig- ly. naling resulted in the loss of cytoskeletal stre- ss fiber formation, which was associated with In contrast to the studies described above, oth- decreased adhesion of cells to fibronectin and ers research has shown that increased expres- diminished protein secretion. Using the same sion of PIP in ZR-75-1 cells caused by andro- experimental approach-breast cancer cell lines gens was associated with their growth arrest with suppressed expression of the PIP gene [74, 99]. In line with these results, recent micro- [36], found that the absence of PIP protein array analysis revealed a significant increase in causes cell cycle arrest in the G1 or mitotic the expression of genes associated with an phase and cytokinesis defect. Furthermore, a anti-proliferative and pro-apoptotic effects in correlation between expression of PIP and cell PIP-expressing breast cancer cells in compari- cycle-related genes, especially those involved son to breast cancer cells with no expression of in mitotic transition was observed. Among ana- PIP [100]. lyzed breast cancer cell lines, T47D and MDA- MB-453 cells with silenced expression of the The silencing of PIP expression in breast can-

PIP gene underwent G1 arrest manifested by a cer cells inhibited their invasive properties

10% to 20% increase in the G0-G1 cell popula- through modulating the integrin signaling path-

2157 Am J Cancer Res 2018;8(11):2150-2164 Urbaniak et al.: PIP in breast cancer way [81]. It was proposed that PIP as asapartyl 10.1007/s00018-008-8463-x. [Cell Mol Life protease increases the invasion of cancer cells Sci.]). Figure 4B Reprinted by permission from: by generating fibronectin peptides, which bind John Wiley and Sons (The Licensor): [Journal to integrin β1. Also, microarray analysis reve- Publisher (John Wiley and Sons)] [JOURNAL aled a significant increase in the expression of NAME: Journal of Cellular Physiology] [REFER- genes associated with migratory properties in ENCE CITATION: (Runx2 controls a feed-for- PIP-expressing breast cancer cells in compari- ward loop between androgen and prolactin- son to breast cancer cells with no expression of induced protein (PIP) in stimulating T47D cell PIP [100]. There is also some evidence that PIP proliferation, Sanjeev K. Baniwal, Gillian H. is involved in cell-cell and cell-matrix (fibronec- Little, Nyam-Osor Chimge, et al.), [COPYRIG- tin) adhesion of breast cancer cells [101]. HT] (2012), 2012 May; 227 (5): 2276-82, (doi: 10.1002/jcp.22966 [J Cell Physiol.]). In breast cancer, an efficient Th1 response is associated with smaller tumor size [102] and Disclosure of conflict of interest better prognosis [103]. Therefore, it was pro- None. posed recently that PIP may play an advanta- geous role during the progression of breast Address correspondence to: Piotr Dziegiel, Depart- cancer, especially during early stages, because ment of Human Morphology, Division of Histology it positively affects differentiation of T lympho- and Embryology, Wroclaw Medical University, Chalu- cytes to CD4 positive Th1-cells [104]. However, binskiego 6a, Wroclaw 50-368, Poland. Tel: 71-784- there is lack of direct experimental data sup- 13-55; Fax: 71-784-00-82; E-mail: piotr.dziegiel@ porting such a hypothesis. umed.wroc.pl; Maciej Ugorski, Laboratory of Glyco- biology, Ludwik Hirszfeld Institute of Immunology Conclusions and Experimental Therapy, Polish Academy of Sci- ences, Wroclaw, Poland. E-mail: [email protected]. PIP is produced and excreted by normal cells wroc.pl and tissues characterized by apocrine-type se- cretion. However, it is also found in large am- References ounts in some pathological conditions such as cystic fluid from mastopathy and some cancer [1] Haagensen DE, Mazoujian G, Dilley WG, Peder- cells, especially breast cancer cells. PIP plays sen CE, Kister SJ and Wells SA. Breast gross a role in cell proliferation, migration and adhe- cystic disease fluid analysis. I. Isolation and sion, and is generally described as a protein radioimmunoassay for a major component pro- tein. J Natl Cancer Inst 1979; 62: 239-247. with immunomodulatory properties. However, [2] Haagensen DE, Gall SA, Brazy JE, Giannola J its functions in both cancerous and normal and Wells SA. Analysis of amniotic fluid, ma- cells have not yet been fully elucidated and in ternal plasma, and cord blood for a human many aspects are controversial. Therefore, fur- breast gross cystic disease fluid protein. Am J ther studies regarding this glycoprotein, which Obstet Gynecol 1980; 138: 25-32. is undoubtedly important for cell functioning [3] Shiu RP and Iwasiow BM. Prolactin-inducible are still necessary. proteins in human breast cancer cells. J Biol Chem 1985; 260: 11307-11313. Acknowledgements [4] Murphy LC, Tsuyuki D, Myal Y and Shiu RP. Iso- lation and sequencing of a cDNA clone for a “The project was financed from the funds of prolactin-inducible protein (PIP). Regulation of the National Science Center granted on the PIP gene expression in the human breast can- basis of decision No. DEC-2016/23/B/N25/ cer cell line, T-47D. J Biol Chem 1987; 262: 02647”. Figure 2A Reprinted by permission 15236-15241. from Springer Nature (The Licensor): [Journal [5] Myal Y, Gregory C, Wang H, Hamerton JL and Shiu RP. The gene for prolactin-inducible pro- Publisher (Springer)] [JOURNAL NAME: Cellular tein (PIP), uniquely expressed in exocrine or- and Molecular Life Sciences] [REFERENCE gans, maps to chromosome 7. Somat Cell Mol CITATION: (Prolactin inducible protein in can- Genet 1989; 15: 265-270. cer, fertility and immunoregulation: structure, [6] Akiyama K and Kimura H. Isolation of a new function and its clinical implications, Md. I. actin-binding protein from human seminal Hassan, A. Waheed, S. Yadav et al.), [COPY- plasma. Biochim Biophys Acta 1990; 1040: RIGHT] (2008), 2009 Feb; 66 (3): 447-59, (doi: 206-210.

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