Aberrant Subcellular Immunolocalization of NOTCH-1 Activated Intracellular Domain in Feline Mammary Tumours

J. Comp. Path. 2014, Vol. 150, 366e372

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NEOPLASTIC DISEASE

Aberrant Subcellular Immunolocalization of NOTCH-1 Activated Intracellular Domain in Feline Mammary Tumours L. Ressel*,‡, R. W. Else†, A. Poli‡ and D. J. Argyle† * Veterinary Pathology, School of Veterinary Science, University of Liverpool, Leahurst Campus, Neston CH64 7TE, † Royal (Dick) School of Veterinary Studies and Roslin Institute, Easter Bush Veterinary, Roslin, Midlothian EH25 9RG, UK and ‡ Department of Animal Pathology, Faculty of Veterinary Medicine, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy

Summary NOTCH-1 is a transmembrane receptor protein. Ligand proteins expressed on the surface of neighbouring cells bind to the NOTCH-1 extracellular domain by juxtacrine signalling and release the NOTCH intracellular domain (NICD) to alter gene expression. Forty feline mammary lesions (34 malignant and six hyperplastic) were submitted for immunohistochemical analysis of NICD expression using an anti-feline NICD monoclonal antibody. Associations between NICD expression in carcinomas and morphological parameters, as well as overall survival (OS), were investigated. NICD nuclear expression was observed in hyperplastic lesions (100%) while cytoplasmic localization was evident in carcinomas (0% nuclear positive; 87.5% cytoplasmic positive; 12.5% negative). Cytoplasmic NICD localization was statistically associated with carcinomas, while nuclear labelling was associated with hyperplasia. No significant correlation between positive or negative NICD expression and OS or morphological parameters was detected. NOTCH-1 activation, immunohistochemically identified by the NICD active form, appears to play a role in feline mammary carcinoma biology as the majority of tumours express this protein. Nuclear localization is consistent with the established NICD metabolic intranuclear pathway while cytoplasmic accumulation suggests aberrant NOTCH-1 signalling typical of malignant tumour progression. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: cat; mammary tumours; NOTCH-1; NOTCH-1 intracellular domain

Introduction NOTCH-1 (also known as TAN-1) is a transmembrane receptor. Ligand proteins such as JAG-1, JAG-2, DLL1, DLL2 and DLL3 (Cohen et al., 2010) expressed on the surface of neighbouring cells bind to the NOTCH-1 extracellular domain (juxtacrine signalling mechanism) inducing a gamma secretase-dependent proteolytic cleavage of the protein, leading to the release of the NOTCH intracellular domain (NICD) into the cytoplasm. NICD then enters the cell nucleus to alter gene expression. NOTCH-1 plays a key role in a wide range of mechanisms involved in cellular differentiation such as neoCorrespondence to: L. Ressel (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.11.213

angiogenesis (Ranganathan et al., 2011), haematopoiesis (Pajcini et al., 2011) and maturation of several organs and tissues including the prostate, smooth muscle (Wu et al., 2011) and mammary gland epithelium (Raafat et al., 2011). NOTCH-1 plays a role in many different human tumours including those of the brain (Jiang et al., 2011), skin, kidney (Sjolund et al., 2008) and breast (Wu et al., 2007). In tumour biology it has been demonstrated that NOTCH-1 influences epidermal growth factor (EGF) receptor pathways (Xu et al., 2010), phosphate and tensin homologue (PTEN) (Shou et al., 2001; Whelan et al., 2009), angiogenesis (Shi and Harris, 2006) and epithelial-to-mesenchymal transition (EMT) (Ranganathan et al., 2011). Recently, it has also been shown that the role of NOTCH-1 is Ó 2013 Elsevier Ltd. All rights reserved.

Expression of NICD in Feline Mammary Tumours

somewhat equivocal; in some contexts NOTCH-1 seems to promote differentiation, proliferation or survival, while in others, NOTCH-1 inhibits these processes (Ranganathan et al., 2011). Aberrant activation of NOTCH-1 signalling has been shown in one study to increase the self-renewal capacity of normal mammary stem cells, leading to a tenfold increase in mammosphere formation (Grudzien et al., 2010). Moreover, breast cancer stem cell populations show an up-regulation of NOTCH-1 gene expression (Farnie et al., 2007; Farnie and Clarke, 2007). The role of NOTCH-1 in human breast carcinomas has been investigated extensively (Reedijk et al., 2005). An active NOTCH-1 signalling pathway is often identified as a major escape/survival pathway for cancer cells that are challenged by different therapeutic approaches. NOTCH-1 is up-regulated in neoplastic cells that fail to respond to treatments such as radiotherapy (Wang et al., 2010) or molecular target therapy (Ranganathan et al., 2011). More specifically it has been demonstrated that in TrastuzumabÒ-treated, HER-2-positive or TamoxifenÒ-treated oestrogen receptor (OR)-positive mammary tumours, the NOTCH-1 pathway is initially turned off, but appears to be activated after a longer period of chemotherapy (Osipo et al., 2008). The expression of NOTCH-1 in animals has been investigated in laboratory mouse models of mammary gland tumours (Hu et al., 2006; Raafat et al., 2011) and in primary cell cultures of rodent epithelial cells. In the latter model, NOTCH-1 activation was capable of inducing neoplastic transformation in cooperation with E1A adenovirus infection (Capobianco et al., 1997). At present, no data are available on the expression of NOTCH-1 in feline mammary tissues and feline mammary carcinomas (FMCs); therefore, the aim of the present study was to describe the immunohistochemical expression of active NOTCH-1, represented by NICD, in normal, hyperplastic and neoplastic feline mammary tissues and to investigate any potential correlation with clinicopathological parameters in FMCs.

Materials and Methods Forty queens, which had undergone surgical treatment (total mastectomy) for clinically suspected mammary tumours at the Department of Veterinary Clinics, School of Veterinary Medicine, University of Pisa, between 2003 and 2010, were enrolled in the study. Two normal mammary glands from healthy queens, sampled during routine necropsy examination, were also collected and submitted for NICD immunohistochemistry (IHC). Specimens were fixed

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for at least 48 h in neutral buffered formalin, processed routinely and sections were stained with haematoxylin and eosin (HE). Lesions were classified according to the World Health Organization (WHO) Histological Classification of Mammary Tumours of the Dog and Cat (Misdorp et al., 1999). Morphological parameters evaluated in feline mammary carcinomas were: morphological subtype (tubular carcinoma, solid carcinoma), tumour grade (Castagnaro et al., 1998; WDC, well-differentiated carcinoma; MDC, moderately differentiated carcinoma; PDC, poorly differentiated carcinoma) and mitotic index (total number of mitotic figures in 10 high-power [
400] fields). Invasion of lymphatic vessels around the carcinomas was also evaluated. For IHC, sections (4 mm) were mounted on Superfrost PlusÒ slides (Thermo Scientific, Menzel GmbH & Co., KG, Braunschweig, Germany) and dried overnight at 37 C. Sections were de-waxed in xylene, dehydrated through a graded series of alcohols and rehydrated in deionized water. Antigen retrieval was performed with an Ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) using an automated, computer controlled, antigen retrieval workbench (MilestoneÒ, Bergamo, Italy) for 15 min at 98 C and cooled at room temperature for 20 min. Endogenous peroxidase was blocked with DAKO RealÒ (Dako, Glostrup, Denmark) for 30 min followed by three washes in 0.05% Tween Tris buffered saline solution (TBST; pH 7.6). The primary antibody (mouse monoclonal anti-human/mouse/feline NICD, clone mN1a, Merck/MilliporeÒ, Darmstadt, Germany) diluted 1 in 200 in TBST was applied for 1 h at 37 C. After three TBST washes, a secondary anti-mouse polymer (Envision PlusÒ, Dako) was added for 30 min at room temperature. Binding was ‘visualized’ by use of 3,30 -diaminobenzidine (Impact DABÒ, Vector Laboratories, Burlingame, California, USA) as chromogen. Anagen hair follicles and epidermal basal cells of feline normal skin were used as positive controls. Negative controls were performed by omitting the primary antibody and replacing it with a murine subclass matched (IgG1) unrelated primary monoclonal antibody. NICD expression was judged positive when the presence of distinct brown cytoplasmic and/or nuclear labelling was detected. Evaluation of NICD expression was semiquantitative, based on the mean number of positively-labelled cells in 10 randomly chosen, non-overlapping, lesion representative highpower fields. A NICD distribution grade was assigned based on the number of positive neoplastic/hyperplastic cells as follows: 0, no positive cells; 1, 1e25% labelled cells; 2, 26e50% labelled cells; 3, 51e75% labelled cells; and 4, 76e100% labelled cells.

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Queens diagnosed with mammary carcinomas (n ¼ 34) were followed for 2 years after surgery. Clinical examination and tumour staging were performed 6, 12, 18 and 24 months after surgery. The presence of distant organ metastases and the recurrence of primary tumours were investigated by clinical and radiographic examinations. The queens that died during the follow-up period were submitted for post-mortem examination and tumour-related death was confirmed or excluded. Animals that died during the follow-up period due to a non-tumour related cause were excluded from the study. Twenty-six cats with mammary carcinomas had a complete followup and comprised the study population for survival analysis at the end of the follow-up period. Statistical analysis was performed using the statistical package SPSS Advanced Statistics 13.0 (SPSS Inc., Chicago, Illinois, USA). A chi-square test was used to investigate the significance of the relationship between NICD protein expression and individual variables. Statistical significance was based on a 5% (0.05) significance level. Overall survival (OS) analysis was performed using the KaplaneMeier method and the TaroneeWare test was used to investigate the relationship between NICD expression and OS.

Results At the time of diagnosis, the mean age  standard deviation of the queens bearing mammary carcinomas was 10  1.8 years (range 7e14 years). Thirty-four lesions were classified as feline mammary carcinomas and six as mammary hyperplasia. No adenomas were diagnosed. The hyperplastic mammary lesions (n ¼ 6) were all classified as lobular hyperplasia. Carcinomas (n ¼ 34) were all of the simple type and no complex carcinomas were diagnosed. Simple carcinomas comprised tubulopapillary (n ¼ 20) or solid (n ¼ 14) subtypes. The Elston and Ellis grading system (Elston and Ellis, 1991) was used to divide tumours into WDCs (n ¼ 10), MDCs (n ¼ 14) or PDCs (n ¼ 10). At the time of diagnosis, invasion of lymphatic vessels around the tumour was present in 16 feline carcinomas (47%). The mitotic index of mammary carcinomas ranged from 2 to 50, with a median of 13.5 and an average of 15.8. In the positive control tissue (normal feline skin) trichoblasts of anagen hair bulbs and basal keratinocytes had multifocal nuclear labelling. Normal feline mammary glands were mostly NICD negative, with rare intranuclear labelling of mammary gland lobular cells. In the overlying skin there was multifocal intranuclear labelling of trichoblasts of the hair

bulbs and the epidermal basal layer, as observed in the external positive control. The majority of mammary lesions investigated (both hyperplastic and neoplastic) expressed NICD protein in either the nucleus or in the cytoplasm. In all hyperplastic lesions the NCID labelling was consistently nuclear (Table 1), evident as homogeneous intense immunolabelling (Fig. 1A). All of the hyperplastic lesions examined exhibited grade 1 immunoreactivity. The majority of FMCs (85.2%) expressed NCID and its immunolocalization was consistently cytoplasmic (Fig. 1B), with focal stippling in the perinuclear area (Fig. 1B). NICD-positive carcinomas had varying percentages of labelled cells, with >50% of the lesions falling into grades 2, 3 or 4 (Table 2). Nuclear expression was statistically associated with hyperplastic lesions, while cytoplasmic localization was statistically associated with carcinomas (P <0.001). Irrespective of the subcellular localization, the percentage of NCID-positive cells was significantly higher (P <0.05) in carcinomas compared with hyperplastic lesions. No statistical association was detected between the number of NICD-positive cells and any of the morphological features of carcinomas investigated (i.e. histological subtype, tumour grade, mitotic index or lymphatic invasion). At the end of the clinical follow-up of the 26 queens with complete clinical information, 15 (62.5%) were still alive, while 11 (37.5%) had died due to mammary carcinomas. There was no significant correlation between positive or negative NICD expression and OS in carcinomas or between OS and different grades of NICD positivity.

Discussion NOTCH-1 activity has been associated with the initiation and progression of neoplastic disease and has been implicated in the maintenance of the neoplastic phenotype and resistance to therapeutic agents, but the function of this gene in tumour biology appears to be strictly context dependent (Ranganathan et al., 2011). In normal mammary gland development Table 1 NICD expression in feline mammary hyperplasia and carcinoma NICD expression Negative

Hyperplasia Carcinoma

0 (0%) 5 (14.8%)

Positive Nucleus

Cytoplasm

6 (100%) 0 (0%)

0 (0%) 29 (85.2%)

Figures shown are number (and relative percentage) of positive lesions.

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Expression of NICD in Feline Mammary Tumours

Fig. 1. Immunohistochemical expression of NICD in lobular hyperplasia and mammary carcinoma. (A) Mammary gland lobular hyperplasia. In benign mammary hyperplastic lesions some epithelial cells show distinct nuclear NICD labelling (inset, arrow) indicative of intranuclear localization of NICD protein. (B) Mammary carcinoma. A large number of carcinoma cells show cytoplasmic NICD expression (inset, arrow) indicative of cytoplasmic localization of NICD protein. IHC. Bars, 50 mm.

NOTCH-1 activation promotes self-renewal in stem cells, while in later stages of development it promotes mammary progenitor cell differentiation towards a myoepithelial cell fate versus an epithelial cell fate (Ranganathan et al., 2011). Based on the results of

this preliminary study, NOTCH-1 activation, identified immunohistochemically by an anti-feline NICD antibody, appears to play a role in feline mammary gland hyperplastic growth (all hyperplastic lesions expressed NICD), but also in aberrant neoplastic mammary gland growth (85.2% of carcinomas were positive for NCID). In support of this result, it has been demonstrated that constitutive activation of NOTCH-1 signalling prevented differentiation of mouse mammary epithelial cells, which led to hyperplastic glandular growth resulting in poorly differentiated carcinomas (Jhappan et al., 1992). This is consistent with the finding of the present study, that a significant increase of NOTCH-1 expression was detected in carcinomas compared with benign hyperplastic lesions. Although there is some evidence of genetic alterations in NOTCH genes in solid tumours, NOTCH1 signalling appears to be crucial in sustaining the growth of many tumours including cancers of the breast, colon, pancreas, prostate and central nervous system. These observations support the hypothesis that NOTCH-1 exerts it effects in solid tumours owing to an aberrant activation of the pathway (Ranganathan et al., 2011). These studies underscore the hypothesis that the outcome of NOTCH-1 signalling in tumorigenesis mostly depends on the temporal and spatial context in a given tissue (Ranganathan et al., 2011). For example, it appears that NOTCH1 can function as a tumour suppressor in pancreatic cancers, in contrast to other tissues (Weijzen et al., 2002). The majority of FMCs are reported to be OR negative (Millanta et al., 2005) and an important role has been attributed to NOTCH-1 in the maintenance of OR-negative tumour status in man, as the growth rate of OR-negative tumours is reduced following treatment with a gamma secretase inhibitor (GSI). This suggests a role for the NOTCH-1 pathway since GSIs suppress NOTCH signalling (Lee et al., 2008). A significant result of the present study was the strikingly different subcellular immunolocalization of NICD expression between benign hyperplastic lesions and mammary carcinomas. Nuclear labelling was observed in all hyperplastic

Table 2 NICD immunohistochemical grade in feline mammary hyperplasia and carcinoma NICD grade

Hyperplasia Carcinoma

0

1

2

3

4

0 (0%) 5 (14.8%)

6 (100%) 6 (17.6%)

0 (0%) 7 (20.6%)

0 (0%) 9 (26.4%)

0 (0%) 7 (20.6%)

Grade: 0, no positive cells; 1, 1e25% positive neoplastic/hyperplastic cells; 2, 26e50% positive neoplastic/hyperplastic cells; 3, 51e75% positive neoplastic/hyperplastic cells; 4, 76e100% positive hyperplastic/neoplastic cells.

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glands examined, while carcinoma cells only exhibited cytoplasmic positivity or, in a minority of cases, lacked immunoreactivity for NICD. Nuclear localization of NICD, detected in hyperplastic lesions, is consistent with interaction between NICD and intranuclear promoter proteins involved in transcriptional activation of cell proliferation, a feature that is characteristic of the established NICD pathway and responsible for the physiological differentiation and growth of the mammary gland. A schematic representation of the established NICD pathway is depicted in Fig. 2. On the other hand, cytoplasmic accumulation of such protein, as identified in the present study in the vast majority of carcinomas, may represent an aberrant NOTCH-1 signalling cascade described for the first time by Aster et al. (1997) and subsequently by Perumalsamy et al. (2009). According to this hypothesis, NICD activity could be initiated by a membrane-anchored form of NICD, which interacts with RICTOR and mTOR proteins, triggering the activation of AKT and consequently stimulating cell survival. More recently it has been demonstrated that in mammary tumours, while nuclear translocation was required for canonical NOTCH-1 gene activation, localization of NICD to the cytoplasm was independent of nuclear translocation and sufficient to up-regulate interleukin (IL)-6 production through IKK alpha/IKK beta and

Fig. 2. Established NOTCH-1 pathway. (A) NOTCH-1 transmembrane protein is activated on the target cell by the interaction with a membrane-bound ligand on a neighbouring cell in a juxtacrine manner. (B) This process leads to cleavage of NICD by the action of gamma-secretase and (C) release of free NICD into the cytoplasm. (D) This active form enters the nucleus and accumulates inside it. NICD binds to a repressor and removes it, promoting transcription by (E) activation of a transcription factor. These molecular events lead to cell proliferation and survival.

Fig. 3. Alternative aberrant NOTCH-1 pathway. (A) NOTCH-1 transmembrane protein on the target cell is activated by the interaction with a membrane-bound ligand on a neighbouring cell in a juxtacrine manner. (B) This process leads to cleavage of NCID by the action of gamma-secretase and (C) release of free NICD into the cytoplasm, which accumulates in the proximity of nuclear membrane. (D) NCID activates Akt in a RICTORemTOR-dependent manner, leading to cell proliferation and survival. (E) NICD stimulates the increase of interleukin (IL)-6 production through activation of IKK alpha/IKK beta and mutated p53.

mutant p53 dependent activation (Jin et al., 2013). A schematic representation of the possible aberrant NOTCH-1 pathway is depicted in Fig. 3. Interestingly, raised serum IL-6 concentration in people with mammary cancer has been shown to be a negative prognostic factor (Knupfer and Preiss, 2007). The results of the present study identified aberrant cytoplasmic localization of NICD in feline mammary carcinomas as observed in the previous study. It should be noted, however, that until a deeper investigation unravels the interactions between different proteins involved in the downstream NOTCH-1 signalling in cats, alternative hypotheses to explain the intracytoplasmic localization of NICD should also be taken into consideration. Accumulation of NICD in the cytoplasm may also represent, for example, decreased degradation of the NICD protein or a pathological accumulation of a defective form of NICD. Even though at present the mechanism leading to the cytoplasmic NICD localization of feline cancer cells is not clear, considering the fact that carcinomas exhibited an increased NICD expression as compared with hyperplastic lesions and taking into consideration the recent published data in regard to a dysregulated cytoplasmic pathway of NICD in human mammary carcinomas, an active role of the NICD protein in feline mammary tumour biology seems to

Expression of NICD in Feline Mammary Tumours

be the most likely hypothesis and further investigations are warranted. NICD expression was not correlated with any of the morphological parameters investigated and did not show any correlation with prognosis. This finding may suggest a role in the initiation of neoplastic transformation in FMCs, rather than a molecular step linked with tumour progression and proliferation. In human medicine, only one study (Yao et al., 2010) has described a positive association between NOTCH-1 expression and poor prognosis, but the antibody used in that study identified all forms of NOTCH-1 (both the inactive membrane and the cleaved active intracellular form), so, from this point of view, a direct comparison cannot be made with the present results. It should also be noted that the majority of feline mammary carcinomas are locally aggressive and potentially metastatic and for this reason a 2year follow-up may not be representative of the real malignant potential of those tumours. This is the first report of NICD protein expression in FMCs. NICD nuclear localization, consistent with the established NOTCH-1 activation pathway, seems to characterize benign feline mammary gland hyperplasia. Increased expression and aberrant cytoplasmic localization of NICD appears to be associated with neoplastic transformation in the feline mammary gland. Although further studies are required, this preliminary investigation shows that cytoplasmic localization of NICD occurs in FMCs and suggests that there could be an aberrant NOTCH-1 pathway in feline malignant mammary tumours, which differs from the established pathway that characterizes mammary hyperplasia and may be involved in FMC initiation.

Acknowledgements The authors would like to thank Dr G. Leeming and A. Malbon for critical revision of the manuscript and N. MacIntyre and the technical staff of the Royal (Dick) School of Veterinary Studies, University of Edinburgh for their technical contribution.

Conflict of Interest Statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

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September 1st, 2013 ½ Received, Accepted, November 28th, 2013