Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis

    Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis Ilanit Boyango, Uri Barash, Liat Fux, Inna Naroditsky, Neta Ilan, Israel Vlodavsky PII: DOI: Reference:

S0945-053X(17)30165-8 doi:10.1016/j.matbio.2017.08.005 MATBIO 1355

To appear in:

Matrix Biology

Received date: Revised date: Accepted date:

20 June 2017 3 August 2017 30 August 2017

Please cite this article as: Boyango, Ilanit, Barash, Uri, Fux, Liat, Naroditsky, Inna, Ilan, Neta, Vlodavsky, Israel, Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis, Matrix Biology (2017), doi:10.1016/j.matbio.2017.08.005

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Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis Ilanit Boyango1, Uri Barash1, Liat Fux1, Inna Naroditsky2, Neta Ilan1, and Israel

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Vlodavsky1*

Cancer and Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine,

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Technion, Haifa 31096, Israel; 2Department of Pathology, Ramabm Health Care Campus,

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Haifa, Israel

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Key words: Heparanase, mammary gland, development, signaling, tumorigenesis

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Conflict of interest: The authors have no potential conflict of interest to declare.

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Authors' contribution: Conception and design: IV, NI Development of methodology: IB, UB, LF, NI Acquisition of data: IB, UB, LF, NI Analysis and interpretation of data: IB, LF, UB, NI, IV, IN Writing, review, and/or revision of the manuscript: IB, NI, IV Study supervision: IV

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To whom correspondence should be addressed: Israel Vlodavsky Cancer and Vascular Biology Research Center Bruce Rappaport Faculty of Medicine Technion, P. O. Box 9649, Haifa 31096, Israel Phone: 972-4-8295410; Fax: 972-4-8523947 E-mail: [email protected]

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Abstract Heparanase is an endoglucuronidase that uniquely cleaves the heparan sulfate side chains of heparan sulfate proteoglycans. This activity ultimately alters the structural integrity of

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the ECM and basement membrane that becomes more prone to cellular invasion by

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metastatic cancer cells and cells of the immune system. In addition, enzymatically inactive heparanase was found to facilitate the proliferation and survival of cancer cells by

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activation of signaling molecules such as Akt, Src, signal transducer and activation of transcription (Stat), and epidermal growth factor receptor. This function is thought to be executed by the C-terminal domain of heparanase (8c), because over expression of this

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domain in cancer cells accelerated signaling cascades and tumor growth. We have used the regulatory elements of the mouse mammary tumor virus (MMTV) to direct the expression

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heparanase and the C-domain (8c) to the mammary gland epithelium of transgenic mice. Here, we report that mammary gland branching morphogenesis is increased in MMTVheparanase and MMTV-8c mice, associating with increased Akt, Stat5 and Src

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phosphorylation. Furthermore, we found that the growth of tumors generated by mouse

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breast cancer cells and the resulting lung metastases are enhanced in MMTV-heparanase mice, thus supporting the notion that heparanase contributed by the tumor

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microenvironment (i.e., normal mammary epithelium) plays a decisive role in tumorigenesis. Remarkably, MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands. Although this occurs at low rates and requires long latency,

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it demonstrates decisively the pro-tumorigenic capacity of heparanase signaling.

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Introduction Branching morphogenesis is a fundamental process in the development of diverse epithelial organs such as the lung, kidney, prostate, salivary, lacrimal, submandibular and

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mammary glands. Unlike other branched organs, much of branching morphogenesis of the

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mammary gland occurs after birth during adolescent. In addition to hormones (i.e., estrogen, progesterone, growth hormone, prolactin), mammary gland development is

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regulated by a variety of growth factors, many of which bind heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPG) expressed by mammary epithelial cells [14]. Interaction with HSPG is thought to protect the bound proteins (i.e., growth factors)

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from degradation, creates a storage depot for later release, regulates diffusion through the tissue, and facilitates the assembly and activity of signaling complexes [4-6]. HSPG-

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growth factor interactions are thus considered to function as molecular switches in the course of development, branching morphogenesis and diseases [4, 7, 8]. By interacting with other macromolecules such as laminin, fibronectin, and collagen, HSPG contribute to

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the structural integrity, self-assembly and insolubility of the extracellular matrix (ECM)

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and basement membrane that underlies epithelial and endothelial cells, thus intimately modulating cell-ECM interactions [6, 9]. More specifically, a considerable body of ECM,

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evidence indicates that mammary gland branching morphogenesis is controlled by the ECM-receptors,

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ECM-degrading

enzymes

[10].

Unlike

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metalloproteinases (MMPs) that cleave the protein backbone of HSPG, heparanase is an endoglucuronidase that uniquely cleaves the HS-side chains of HSPG. This activity

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ultimately alters the structural integrity of the ECM and basement membrane that become more prone to cellular invasion by metastatic cancer cells and cells of the immune system [11, 12]. Moreover, cleavage of HS by heparanase releases a variety of polypeptides that are sequestered in the ECM and cell surface, and converts them into bioactive mediators, ensuring rapid tissue response to local or systemic cues [12]. In addition, studies revealed that heparanase also functions in an enzymatic activityindependent manner. Notably, inactive heparanase was found to facilitate proliferation and survival of cancer cells by activation of signaling molecules such as Akt, Src, signal transducer and activation of transcription (Stat), and epidermal growth factor receptor (EGFR) [13, 14]. This function is thought to be executed by the C-terminal domain of heparanase (8c), because over expression of this domain in cancer cells accelerate signaling cascades and tumor growth [15-17].

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In order to examine the role of heparanase in mammary gland development, and to distinguish between enzymatic activity and signaling aspects of heparanase, we applied the regulatory elements of the mouse mammary tumor virus (MMTV) to direct the expression

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heparanase and its C-domain (8c) to the mammary gland epithelium of transgenic mice.

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Here, we report that mammary gland branching morphogenesis is increased in MMTVheparanase and MMTV-8c mice, associating with increased Akt, Stat5 and Src

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phosphorylation. Furthermore, we found that the growth of tumors generated by mouse breast cancer cells and the resulting lung metastases are enhanced in MMTV-heparanase mice, thus supporting the notion that heparanase contributed by the tumor

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microenvironment (i.e., normal mammary epithelium) plays a decisive role in tumorigenesis [18]. Remarkably, MMTV-8c mice develop spontaneous tumors in their

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mammary gland. Although this occurs at low rates (~15%) and requires long latency (6-10

Materials and methods

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months), it evidently demonstrates the pro-tumorigenic capacity of heparanase signaling.

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Generation of MMTV-heparanase/8c transgenic mice. HindIII-EcoR1 fragment comprising the entire open reading frame of heparanase/8c was cloned into the compatible

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sites in plasmid containing the promoter/enhancer of the mouse mammary tumor virus (MMTV LTR) kindly provided by Dr. Phillip Leder (Harvard Medical School, Boston MA) [19, 20] (Suppl. Fig. 1A). SalI-SpeI linear fragment containing the MMTV-heparanase/8c

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expression cassette was injected into fertilized eggs and transgenic mice were generated essentially as described [21]. Founder mice were back-crossed for 10 generations to reach pure Balb/c background.

Antibodies and reagents. Anti-smooth muscle actin (SMA) and anti-actin monoclonal antibodies were purchased from Sigma (St. Louis, MO); anti phospho-Akt, phospho-Stat5, phosphp-Stat3, and phospho-Src antibodies were purchased from Cell Signaling (Beverly, MA). Anti-Src, anti-Akt, anti-Stat5, anti-phospho-Erk, anti-Erk2 and anti-Ki67 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LYVE antibody was from Abcam (Cambridge, MA); anti-CD45 and anti-cytokeratin 14 antibodies were purchased from Biolegend (San Diego, CA). Rat anti-mouse/human cytokeratin 8 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB; University of Iowa). Anti-heparanase polyclonal antibody (#63) was generated in rabbits immunized with the full-

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length heparanase protein. The heparanase inhibitor PG545 was kindly provided by Zucero Therapeutics (Darra QLD 4076, Australia) [22].

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Histology and immunostaining. 8-weeks old virgin, 13-days pregnant (vaginal plug was

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considered as day 0), and lactation day 1 and day 15 mice were analyzed, as well as involution glands, 3 and 10 days following separation of the pups from their mothers.

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Hematoxylin-eosin staining and immunostaining of 5 micron mammary tissue were carried out as described [16]. Whole-mount staining of mammary glands was performed essentially as described [21]. Briefly, mammary glands were removed, spread onto glass slide and fixed

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in acidic ethanol for 1h, dehydrated through graded ethanol and defated by acetone (18 h, RT). Slides were then rehydrated and stained (18h, RT) with Carmine-Alum (0.2%; Sigma),

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dehydrated in graded ethanol and destained with acetone (18 h, RT). Following incubation in toluene (18 h, RT), slides were mounted and examined under light microscope at low

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magnification.

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Mouse genotyping. The following primer sets were used for genotyping: 8c FTGCATATCATGGAGA CAGACACACTCCTG, 8c R-TTGCTCCTGGTAGGGCCATTC;

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heparanase F- TGCATA TCATGGAGACAG ACACAC TCCTG, heparanase RGAGAATTCTCAGATGCAAGCA GCAACTTTG; GAPDH F- AGAACATCATCCCTGCATCC, GAPDH R- AGCCGTATTCATTGTCATACC. Beta-actin-driven heparanase transgenic (Hpa-

Tg) mice were described previously [21]. In vitro transcription/translation reaction was

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carried out using the TNT kit, according to the manufacturer's (Promega) instructions.

Cells culture and orthotopic tumor models. MDA-MB-231 human breast carcinoma cells were cultured as described previously [23]. EMT-6 and luciferase-labelled 4T1 mouse breast carcinoma cells were kindly provided by Dr. Yuval Shaked (Faculty of Medicine, Technion, Israel) and

Tumor growth was inspected by external measurements or in vivo imaging system (IVIS) [18]; Tumors were removed after two weeks and lung metastases were quantified by counting three weeks thereafter. Tumors were fixed in 4% paraformaldehyde, and subjected to

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immunostaining as described [16]. Immunoblotting of mammary and tumor extracts was

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carried out essentially as described in details elsewhere [16, 26-28].

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Results

MMTV-directed heparanase and its C-terminal domain enhance mammary gland branching morphogenesis. In order to explore the role of heparanase in mammary gland

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development and tumorigenesis we have targeted heparanase and its C-terminal domain (8C; [17]) expression to the mammary gland of transgenic mice utilizing the regulatory

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elements of the mouse mammary tumor virus (MMTV; Suppl. Fig. 1A) [19, 20]. Following validation of mRNA and protein expression by the gene constructs (Suppl. Fig. 1B, C), they were injected to mouse zygotes. We obtained two founder mice of MMTV-heparanase

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(#9 & #10) and one founder mouse of MMTV-8C (8c; Suppl. Fig. 1D), predominantly

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expressing heparanase in the mammary tissue (Suppl. Fig. 1E) and salivary gland (not shown). We first examined the morphology of mammary glands wild-type (WT; Balb/c)

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and MMTV-transgenic mice at different stages of development [i.e., virgin, mid-pregnant (day 13), lactation day 1, and lactation day 15]. Whole mount staining revealed accelerated branching morphogenesis in MMTV-heparanase vs. WT, non-transgenic mice. This effect

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was evident in transgenic strain #9 already at the virgin stage (Fig. 1; Vir, #9), while strain #10 appeared more developed starting at mid-pregnancy (Fig. 1; MP, #10). In addition, the end-buds of MMTV-heparanase at these developmental stages of the mammary gland appeared significantly enlarged (Fig. 1, arrows). A more detailed analysis of mammary gland development was obtained by histological examination (Fig. 2). As noted by the whole-mount staining (Fig. 1), the mammary glands of MMTV-#9 and MMTV-#10 mice appeared more developed at the virgin and mid-pregnant stages, respectively, exhibiting thicker ducts (Fig. 1, #9, Vir) and denser alveolar structures (Fig. 2, Vir, MP; #9, #10). At the first day of lactation, the mammary gland of heparanase (#9 & #10) and MMTV-8C (8c) transgenic mice appeared more developed than WT gland, filling more densely the mammary fat pad (Fig. 2, Lac Day 1). This effect was still evident, albeit less prominently, on day 15 of lactation, when milk production is highest (Fig. 2, lower panel). Mammary gland development was further quantified by measuring the area in each slide filled with

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epithelial structures (ductal and alveolar). The analyses (Suppl. Fig. 2) further revealed a statistically significant 2-4 fold increase of mammary gland development in MMTVheparanase/8c mice.

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Notably, enhancement of the branching morphogenesis resembled the timing of heparanase

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expression, evident by immunostaining, in each of the transgenic strains (Fig. 3). Thus, in strain #9, heparanase staining is already detected at the virgin stage (Fig. 3, upper second

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left panel), in strain #10 heparanase staining is observed starting at mid-pregnancy (Fig. 3, second middle panel), and the 8c variant is being expressed at lactation (Fig. 3, third panel, second right). Closer examination revealed that heparanase staining is also localized within

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the cell nuclei of MMTV-#9, #10, and 8c (Fig. 3, third and fourth rows), location previously correlated with increased cellular differentiation [29, 30]. Importantly, even

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higher expression of MMTV-heparanase was observed during the involution phase of the mammary glands (Fig. 3, lower panels). This was correlated with a delay in involution of the transgenic mammary tissues evident by large alveoli and ductal structures 10 days after

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separation of the pups from their mothers (Suppl. Fig. 3, right panels, arrows). Notably, the

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increased branching morphogenesis and delayed involution in the MMTV-heparanase mice mimics the phenotype described previously for transgenic mice in which heparanase

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expression was driven by the beta-actin promoter [21]. Unlike MMTV, the actin promoter drives transgene expression in essentially all cell types and tissues to high levels, and a resulting phenotype may be indirect due to systemic factors rather than local effect. The above results clearly show that specific targeting of heparanase or the C-domain to the

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mammary epithelium at relatively low levels (Fig. 3, right panels vs. #9, #10, 8c) is sufficient to enhance mammary gland development.

Enhanced signaling pathways in MMTV-heparanase/8c mammary glands. In order to reveal the molecular mechanism underlying the increase in mammary development we examined the phosphorylation levels of Stat5 implicated mostly in proliferation of the mammary epithelium during pregnancy and lactation and milk protein gene expression [31]. Immunostaining revealed that Stat5 phosphorylation was noticeably increased in MMTV-#9 mice during pregnancy and lactation compared with WT non-transgenic littermates (Fig. 4A, #9 vs. WT), in agreement with previous results tying heparanase and Stats phosphorylation [27]. Increased Stat5 phosphorylation was further confirmed by immunoblotting and appeared comparable in all transgenic strains (Fig. 4B). Furthermore,

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Akt phosphorylation was increased noticeably in MMTV-heparanase (#9) and Hpa-Tg mice but not in MMTV-8c mice (Fig. 4C, upper panel), suggesting that Akt phosphorylation levels do not tie with mammary development. Notably, however, Src

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phosphorylation was markedly increased in all the transgenic strains (Fig. 4C, third panel),

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suggesting that Src phosphorylation may compensate for the lack of Akt activation (Fig. 4C, 8c) in driving mammary gland development. Taken together, these results strongly

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suggest that heparanase signaling properties initiated by its C-domain (8c) enhances mammary gland development.

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Stromal heparanase augments tumor growth and metastasis. We have reported recently that tumor growth is enhanced by heparanase contributed by the tumor microenvironment

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[18, 32]. In order to further reveal the significance of stromal heparanase for tumor growth we implanted EMT-6 mouse breast carcinoma cells in the mammary tissue of virgin WT and MMTV-heparanase (#9) mice. We first established that this model system is

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heparanase-dependent because tumor growth was attenuated markedly by the heparanase

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inhibitor, PG545 (Suppl. Fig. 4A). This inhibitor has proven efficacious in different models of breast cancer [33, 34]. We next implanted EMT-6 cells in the mammary gland of WT

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and MMTV-heparanase/8c virgin mice and tumor growth was inspected. Notably, EMT-6 cells implanted in MMTV-#9 mice grew bigger in volume (Suppl. Fig. 4B) and weight (Suppl. Fig. 4C) compared with WT mice, associating with enhanced Src phosphorylation

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(Suppl. Fig. 4D). Tumor growth in MMTV-#10 and 8c mice was indifferent vs. WT mice (Suppl. Fig. 4B). This agrees with the finding that only MMTV-#9 mice express heparanase in the virgin mammary gland (Fig. 3). We subsequently implanted luciferaselabelled 4T1 mouse breast carcinoma cells in the mammary glands of WT and MMTV-#9 virgin female mice and tumor growth was inspected by IVIS. We found that tumor growth was increased three-folds in MMTV-#9 vs. WT mice (Fig. 5A), differences that are statistically highly significant (p=0.016). Moreover, spontaneous metastasis of 4T1 cells to the lungs was increased two-folds in MMTV-#9 vs. WT mice (Fig. 5B, left; p=0.012). These results strongly imply that heparanase contributed by the stroma (i.e., normal mammary epithelium) enhances tumor growth and metastasis. In addition, heparanase derived from the normal mammary epithelium appears to shape the composition of the tumor microenvironment. This is concluded because immunostaining showed increased number of intra-tumor lymph vessels in MMTV-#9 vs. WT tumors (Fig. 5C, left upper and

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middle panels), and tumor cells were clearly evident inside lymph vessels developed in MMTV-#9 (Fig. 5C, arrow, left lower panel) but not in WT lymphatics (Fig. 5C, upper left). Moreover, 4T1 tumors developed in MMTV-#9 mice were more densely decorated

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with SMA-positive cells compared with WT mice (Fig. 5C, right upper and middle panels).

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Spontaneous tumor development in MMTV-8c mice. Despite the observed enhancement of Stat5, Src, and Akt phosphorylation (Fig. 4), increased mammary development [21], and delayed involution (Suppl. Fig. 2), Hpa-Tg mice do not develop spontaneous tumor lesions during the life span of a mouse, or during breeding. Unexpectedly, the MMTV-8c mice

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were noted to develop tumor lesions spontaneously in their mammary gland (Fig. 6A, upper middle panel), at a low rate (~15%) and after long latency period (6-10 months).

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These lesions do not develop in virgin females but rather in female mice that went through at least one pregnancy, in accordance with earlier reports [20]. The tumors showed morphology typical of invasive breast carcinoma (Fig. 6A, upper left panel), stained

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positive for cytokeratin 8 (CK8; Fig. 6A, second middle panel), were endowed with strong

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staining for heparanase (Fig. 6, left second panel), and exhibited high proliferative capacity (Ki67; Fig. 6, left third panel) associated with Stat3 (Fig. 6, fourth left), Erk and Akt

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phosphorylation (Fig. 6, left fifth and lower panels). In addition to mammary tumors, these mice also developed tumors in the neck region, characterized as invasive salivary gland carcinoma (Fig. 6, right panels) and exhibiting signaling characteristics comparable with

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the mammary carcinoma (Fig. 6, right panels). This pattern of tumor development is typical of the MMTV promoter [19, 20, 35]. Furthermore, some MMTV-8c mice also developed hematological lesions in their abdomen (Suppl. Fig. 5A, left panels), with massive infiltration to the lung (not shown) and kidney (Suppl. Fig. 5A, lower left). The hematological nature of these lesions is concluded by histological analysis and positive staining for CD45 (Suppl. Fig. 5A, left, CD45), possibly implying the induction of additional oncogene(s) such as Myc or Ras [19, 36]. Indeed, high Myc expression was detected in MMTV-8c tumors (Fig. 6B), possibly implying co-operation between heparanase 8c and Myc in the course of spontaneous mammary carcinogenesis.

Discussion Breast cancer is the most common type of cancer in women. Despite major advances in its diagnosis and treatment, several major clinical and scientific questions remain unsolved. It

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is therefore of critical importance to identify genetic alterations responsible for the initiation and promotion of malignant transformation of the breast epithelium in order to identify targets for novel drug discovery. In mouse models of human breast cancer, the

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MMTV-LTR promoter has most frequently been used, and transgenic models of breast

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cancer have provided invaluable insights into the molecular mechanisms of breast carcinogenesis and tumor progression [36]. Importantly, the genetically engineered mouse

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models often develop breast carcinomas that are indistinguishable from human neoplasia [36], thus strengthening the clinical relevance of these models.

Here, we show for the first time that MMTV-8c mice develop spontaneous invasive

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carcinomas in their mammary and salivary glands (Fig. 6). This was unexpected given that transgenic mice in which heparanase expression is guided by the beta-actin promoter [21]

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did not ever develop tumors spontaneously during nearly 15 years of follow-up and dozens of inspected virgin and breeding female and male mice. Tumor development in MMTV-8c mice occurred only in breeding females, which is typical of MMTV-driven tumors [20], in

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accordance with the regulation of the MMTV promoter by steroid hormones [36], and

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required a latency period of 6-10 months. Even longer latency period (18-22 months) was required for tumor development in MMTV-cyclin D1 mice [37], suggesting that additional

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genetic changes are obligatory for the development of overt mammary carcinoma. This is best exemplified by breeding MMTV-Myc and MMTV-Ras mice, resulting in mammary tumors with a dramatically shortened latency period compared with each transgenic mouse strain alone [19]. Heparanase, therefore, does not seem to function as an oncogene and

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cannot elicit malignant transformation on its own, but rather co-operates with cellular oncogenes to promote tumor initiation and growth. This is best demonstrated by cooperation of heparanase or its C-domain with Ras in promoting skin and breast cancer [16]. Breeding of our MMTV-8c with MMTV-Ras or MMTV-Myc (which appears to be induced in MMTV-8c mice; Fig. 6B) mice is expected to resolve this possibility. These studies are currently ongoing. The MMTV-8c tumors are diagnosed as invasive carcinoma, show high cell proliferation capacity (Ki67, Fig. 6A, left) associated with positive staining for phospho-Stat3, phosphoErk and phospho-Akt (Fig. 6, left panels), thus further signifying the capacity of the 8c domain to promote signaling. Similar characteristics were also found in the salivary carcinomas developed in MMTV-8c mice (Fig. 6, right), in agreement with a strong signaling aspect of heparanase in head and neck carcinoma cells [26, 27, 38]. Enhanced Akt, Erk, Stat3 and EGFR phosphorylation is also evident in Ras-mutant MDA-MB-231

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human breast carcinoma cells [39] over-expressing the 8c domain (Suppl. Fig. 5B) and in Ras-transformed MCF10AT1 cells [16], yet the full repertoire of the heparanase 8c domain awaits in-depth appreciation. Taken together, these results support and further expand the

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notion that heparanase is intimately engaged in promoting signaling cascades and

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providing, in part, a molecular mechanism underlying the pro-tumorigenic function of heparanase in breast cancer [40]. More specifically, the results encourage the development

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of compounds that will target the signaling properties of heparanase. Such compounds, together with inhibitors of heparanase enzymatic activity [12, 22], are expected to neutralize the pro-tumorigenic capacity of heparanase in a much broader terms.

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Specific targeting of heparanase or the 8c variant to the mammary epithelium at relatively low levels (Fig. 3, Tg vs. #9, #10, 8c) is sufficient to enhance mammary gland

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development evident by larger end-buds (Fig. 1, arrows) and more alveolar structures that densely fill the mammary fat pad (Fig. 2; Suppl. Fig. 2). This is in agreement with enhanced branching morphogenesis shown previously in the mammary gland of Hpa-Tg

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mice [21] and explants [41]. Enhanced branching morphogenesis in MMTV-8c mice

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clearly implies that signaling aspects of heparanase play a role in mammary gland development, in addition to its enzymatic aspect and cross-talk with MMP-14 [41]. More

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specifically, Stat5 phosphorylation was enhanced significantly in the mammary glands of all heparanase/8c transgenic strains (Fig. 4A, B), further tying heparanase levels with Stat(s) phosphorylation [27]. Notably, phosphorylation of Src was enhanced markedly in all transgenic strains, closely associating with mammary gland development (Fig. 4). This

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confirms and further expands the close association between heparanase and Src phosphorylation in cancer [26, 27, 42-44] and development. Mechanistically, Src and related family members have been shown to enhance the signaling initiated by many growth factors including prolactin, leading to augmented Stat5 activation [45]. Such a scenario may explain the increase in Stat5 phosphorylation levels in mid-pregnant and lactating mammary glands of MMTV-heparanase/8c and Hpa-Tg mice (Fig. 4). Src is also implicated in cellular transformation of mammary cells by, among others, MMTV [46], and increased Src activity is often found in human breast carcinomas [47], associating with eminent Stat3 activation [48]. Augmented Src phosphorylation was also noted in tumors developed by EMT6 and 4T1 cells implanted in MMTV-#9 mice (Fig. 5; Suppl. Fig. 4). This apparently places Src phosphorylation as a key determinant in heparanase/8c signaling that drives mammary development and tumorigenesis.

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High levels of heparanase/8c expression are maintained during the involution phase of the mammary gland (Fig. 3, lowest panels), associating with delayed involution evident by larger alveolar and ductal structures at day 10 of involution compared with WT mice

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(Suppl. Fig. 3, right). Experiments using a variety of rodent models revealed that delayed

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involution often associates with enhanced tumor formation [49]. The transcriptional profile of involuting mammary gland exhibits high level of similarity to that found in wound

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healing and the tumor microenvironment, including expression of many growth factors, cytokines, and proteases [49]. Whether and how this microenvironment co-operates with the 8c domain to promote tumor initiation and growth is yet to be resolved. Notably,

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MMTV infection delayed involution-induced apoptosis in the mouse mammary gland [46], possibly connecting increased Src activation noted in our MMTV-heparanase/Hpa-Tg mice

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(Fig. 4) with delayed involution (Suppl. Fig. 3).

The exceptional early expression of heparanase in virgin female #9 mice (Figs 1-3) enabled us to examine the significance of host heparanase in tumor growth. Implantation of 4T1-

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luciferase cells in virgin #9 mice resulted in tumors that were three-times larger than

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tumors developed in WT mice (Fig. 5), thus strongly supporting the notion that heparanase contributed by the host (i.e., normal mammary epithelium) enhances tumor growth [18,

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32]. Furthermore, not only tumor growth but also spontaneous lung metastasis was increased in #9 mice, associating with alterations of the tumor microenvironment (Fig. 5). This appears significant given the fundamental role that cancer-associated fibroblasts (CAF) play in breast cancer growth and metastasis [50]. Thus, targeting tumor heparanase

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and heparanase originated from the tumor microenvironment as well as heparanase signaling is expected to have a profound effect on tumor growth and metastasis.

Acknowledgments This study was supported by research grants awarded to I.V. by the Israel Science Foundation (grant 601/14); the United States-Israel Binational Science Foundation (BSF); and the Israel Cancer Research Fund (ICRF); I. Vlodavsky is a Research Professor of the ICRF.

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Figure legends

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Figure 1. MMTV-heparanase transgenic mice exhibit increased mammary glands branching morphogenesis. Whole mount staining of mammary gland at different stages of

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development is shown for WT control and MMTV-heparanase strains #9 and #10 mice. Note, increased branching of virgin and mid-pregnant MMTV-heparanase strains #9 and #10, respectively, compared with control non-transgenic littermates. Original

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magnification: x10.

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Figure 2. Histological examination of MMTV-heparanase transgenic mice show more developed mammary glands. Mammary tissues were collected from MMTV-heparanase/8c mice at the indicated stage, fixed with formalin and embedded in paraffin and five micron

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sections were stained with hematoxylin and eosin. Vir- virgin females at 8 weeks of age;

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MP- day 13 of pregnancy (vaginal plug considered time zero); Lac day1- one day following birth; Lac day 15- fifteen days after birth. Original magnification: x25. Note

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increased mammary gland branching morphogenesis of MMTV-heparanase/8c transgenic mice compared with WT non-transgenic littermates.

Figure 3. Heparanase immunostaining correlates with mammary glands morphogenesis.

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Five-micron sections of mammary tissues collected from WT control and MMTVheparanase/8c transgenic mice at the indicated developmental stage were subjected to immunostaining applying anti-heparanase antibody. Original magnification: x100; fourth panel x200. Figure 4. MMTV-heparanase mammary glands show enhanced Stat5 and Src phosphorylation. A, B. Stat5 phosphorylation. A. Immunostaining. Mammary tissue was harvested from WT control and MMTV-heparanase (#9) mice at the indicated developmental stage and 5 micron sections were subjected to immunostaining applying anti-phospho-Stat5 antibody. B. Immunoblotting. Mammary tissue was harvested from WT and MMTV-heparanase transgenic strains (8c, #9, #10) at day 1 of lactation and lysate samples were subjected to immunoblotting applying antibodies directed against phosphoStat5 (upper panel) and Stat5 (second panel). Quantification of Stat5 phosphorylation is

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shown graphically at the lower panel. C. Mammary lysates from the indicated mouse strain were similarly subjected to immunoblotting applying anti-phospho-Akt (pAkt, upper panel), anti-Akt (second panel), anti-phospho-Src (pSrc, third panel), anti-Src (fourth

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panel) and anti-actin (lower panel) antibodies. Figure 5. Tumors grow bigger in MMTV-heparanase (#9) mice. A. tumor growth. 4T1-

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luciferase cells (5x104) were implanted orthotopically in the mammary glands of 8-weeks old WT and #9 mice and tumor growth was inspected by IVIS. Shown is tumor luminescence two weeks after cell implantation (A); quantification of the luminescence is

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shown graphically at the lower panel. B. Tumor metastasis. The mammary tumors were removed and lung metastases were quantified three weeks thereafter. Representative

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images of lung metastases (arrows) are shown in the right panel. C. Immunostaining. Fivemicron sections of 4T1 tumors developed in WT (upper panels) and #9 (middle and lower panels) mice were subjected to immunostaining applying anti-LYVE (a marker for

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lymphatic endothelial cells) and anti-SMA antibodies. Note penetration of tumor cells into

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lymph vessels (lower left, white arrow) and a significant increase in SMA-positive cells in #9 tumors. Most SMA-positive cells are likely fibroblasts because SMA staining was

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rarely observed in the context of blood vessels (lower right, black arrow). Original magnifications: upper and middle left x100, lower left x200; upper and middle right x50, lower right x100. HM: high magnification. D. Extracts of tumors developed in WT and #9MMTV mice were subjected to immunoblotting applying phospho-Akt (upper panel),

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phospho-Src (second panel), Src (third panel), phospho-Erk (fourth panel) and Erk (lower panel) antibodies.

Figure 6. MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands. Tumors developed spontaneously (middle upper panel) in the mammary (left panels) or salivary (right panels) glands were fixed in formalin and embedded in paraffin. Five-micron sections were subjected to histological examination following staining with hematoxylin and eosin (upper panels), or to immunostaining applying anti-heparanase (second panels), anti-Ki67 (third panels), anti-phospho-Stat3 (pStat3; fourth panels), antiphospho-Erk (fifth panels) and anti-phospho-Akt (lower panels) antibodies. Note high cell proliferation (Ki67), Stat3, Erk and Akt phosphorylation levels in tumors developed spontaneously in MMTV-8c mice. Immunostaining for cytokeratin 8 (CK8) is shown in the second middle panel. Original magnifications: x100. B. Myc expression. Total RNA

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was extracted from control mammary gland (MG CONT) and two tumors that were developed spontaneously in the mammary gland of MMTV-8c mice (MG TU1, MG TU2). Corresponding cDNAs were subjected to quantitative real-time PCR analyses applying

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primer set specific for c-Myc. Note a marked increase of Myc levels in the spontaneous

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tumors developed in MMTV-8c mice, also evident by strong immunostaining for Myc

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Highlights We applied the MMTV promoter to direct the expression of heparanase or its c-terminus signaling domain (8c) to the mammary gland of transgenic mice.

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Mammary gland branching morphogenesis is enhanced in MMTV-heparanase/8c mice, associating with increased Akt, Stat5 and Src phosphorylation and signifying the involvement of heparanase signaling capacity.

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Growth of mammary tumor xenografts is enhanced in MMTV-heparanase mice, indicating that heparanase contributed by the tumor microenvironment plays a decisive role in tumorigenesis.

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MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands, emphasizing the significance of heparanase signaling properties in tumorigenesis.