The related protein 2 (SFRP2) in canine mammary

The top upregulated genes among
malignant and benign tumors in our study were found to have a significant role
in cancer progression and tumor invasiveness, as evidenced by human cancer
studies. Top five most upregulated genes in malignant
tumors were COL11A1, SFRP2, LCN2, COL2A1 & H19. COL11A1
gene encodes one of the two alpha chains of type XI collagen, a minor fibrillar
collagen
which had been implicated in tumor
progression in humans. Overexpression of COL11A1 has been reported in
mesenchymal derived tumors (Fisher et al., 2001, Wang et al., 2001, Xu et al.,
2003, Sok et al., 2003, Barnei et al., 2006, Chong et al, 2006, Pilarsky et
al., 2008).  Overexpression is correlated
with poor clinical outcome in human ovarian cancer and advanced stages of
cancers. Wu et al., reported that COL11A1 may promote tumor aggressiveness via
the TGF-?1 and MMP3 axis (Wu et al., 2014). Halstead et al. (2008)
reported expression of COL11A1 in epithelial cells, stroma, and vessels of
normal and cancerous breast tissue (Halstead et al., 2008).

            Secreted
frizzled-related proteins (SFRPs), constitute a family of 5 members (SFRP 1–5)
that modulate Wnt signaling. These proteins are presumed extracellular Wnt
inhibitors.  Overexpression of secreted
frizzled related protein 2 (SFRP2) in
canine mammary gland tumors has been shown to induce cancerous transformation in normal mammary epithelial
cells (Lee
et al. 2004). SFRP2 is localized in the extracellular matrix of mammary gland
tumors. The anti-apoptotic function of SFRP2 is mediated through activation of NF-?B
or Janus kinase (JNK) suppression (Lee et al., 2006). Further, in vitro, as well as, in vivo oncogenic potential of SFRP2 has been demonstrated in
renal cancer. Recently,
SFRP2 has been related to poor prognosis along with genes associated
with epithelial-to-mesenchymal transition (Vincent et al., 2017). Overexpression of SFRP2 was seen in both malignant
and benign mammary tumors in our experiment.

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            Lipocalin (LCN2), another top upregultaed gene among
malignant tumors in our data set, has been shown to be associated with estrogen
receptor (ER)-negative breast tumors in humans (Gruvberger et al., 2001). The
gene is significantly increased in the luminal epithelial cells compared with
myoepithelial cells (Jones et al., 2004), an important finding because the
majority of breast carcinomas are thought to arise from the luminal epithelial cells
(Rakha et al., 2008). LCN2
promotes breast cancer progression by inducing epithelial to mesenchymal
transition (EMT) through the ER?/Slug axis (Yang et al., 2009). LCN2 also plays
an important role in promoting cell migration and invasion of prostate cancer
by inducing epithelial to mesenchymal transition through the ERK/SLUG axis
(Ding et al., 2015). Recently
it has been shown to be associated with tumor invasiveness of human cervical
cancer (Chung et al.,
2016)

Ganpathi et
al., (2016) revealed that high expression of collagen type II, alpha 1 (COL2A1)
was associated with delayed time to recurrence in high grade serious ovarian
cancers. COL2A1 gene has been reported to undergo
somatic alterations in cases of  chondrosarcoma and enchondroma cases (Totoki
et al., 2014).

H19 is a gene for a long noncoding RNA,
having a role in the negative regulation of body weight and cell proliferation. Numerous studies have reported
H19, to have key regulatory functions in tumor development and progression (Brannan et
al., 1990). Dugimont
(1995) reported expression of H19 gene in both epithelial and stromal
components of human invasive adenocarcinomas (Dugimont et al., 1995). H19
levels have also been reported to be remarkably increased in bladder cancer
tissues. H19 promotes bladder cancer cell migration in vitro and in
vivo. H19 is associated with enhancer of EZH2, and this association results
in Wnt/?-catenin activation and subsequent downregulation of E-cadherin. H19 has
been reported to enhance bladder
cancer metastasis by associating with EZH2 and inhibiting E-cad expression (Luo
et al., 2013). Recently biological and clinical relevance of H19 in Colorectal
cancer patients have been thoroughly covered by   Schwarzenbach, 2016

The most upregulated genes among benign mammary
tumors in our experiment were MMP3, MMP1, AREG, PTHLH and SFRP2. Matrix
metalloproteinases(MMPs) play role in cancer progression by degrading
extracellular matrix and basement membrane and are the main proteolytic enzymes
involved in cancer invasion and metastasis (Stamenkovic, 2000). MMP3 and MMP1
have a synergistic effect on breast cancer carcinogenesis (Padala et al., 2017).
MMP1 is the most widely expressed collagenase and plays role in degradation of
collagen I, II and III. MMP-3 has a high proteolytic efficiency and activates a
number of proMMPs (Visse et al., 2003; Lui et al., 2012).

Amphiregulin (AREG) is one of the many ligands for epithelial
growth factor receptor (Luetteke et al., 1999). It plays a central role in
mammary gland development and branching morphogenesis in organs and is
expressed both in healthy and cancerous tissues (Plowman et al., 1990, Ciarloni
et al., 2007, Kariagina et al., 2010, McBryan et al., 2008). Various studies
have highlighted the functional role of AREG in several aspects of
tumorigenesis, including self-sufficiency in generating growth signals,
limitless replicative potential, tissue invasion and metastasis, angiogenesis,
and resistance to apoptosis (Kariagina et al., 2010). The oncogenic activity of
AREG has already been described in the most common human epithelial
malignancies, such as lung, breast, colorectal, ovary and prostate carcinomas,
as well as, in some hematological and mesenchymal cancers (Berasain et al.,
2007, Ishikawa et al., 2005, Castillo et al, 2006, Johnson et al., 1992, Turring
et al., 1998). Furthermore, AREG is also present in the tumor microenvironment (TME)
and contributes to therapeutic resistance to
several cancer treatments (Ishikawa et al., 2005, Eckstein et al., 2008, Xu et
al 2016).

Parathyroid hormone like hormone
(PTHLH) has previously been reported to be produced by tumor cells in the bone
microenvironment and is implicated in osteoclastic activity and contribute to
osteolytic bone metastasis (Broadus
et al., 1988). Ghoussaini and colleagues combined several datasets for a
genome wide analysis and identified PTHLH as loci for susceptibility for breast
cancer (Ghoussaini et al., 2012). Researchers suggest that PTHLP powerfully
promotes tumor formation in breast cancer (Li et al., 2011).

Impact analysis of major upstream regulators

In our study we identified numerous dysregulated
upstream regulators in both malignant and benign tumors of mammary gland, which
affected expression patterns of various other genes related with tumorogeneis. The
top upstream regulators in malignant mammary tumors were secreted
phosphoprotein (SPP1) and coagulation factor II (F2). Pathway analysis
revealed that secreted phosphoprotein (SPP1) overexpression affected the expression levels of breast cancer type
1 susceptibility protein
(BRCA1) & Cell-division cycle protein 20 (CDC20). BRCA1 is a transcription regulator implicated in
breast cancer pathogenesis and have
been associated with increased risks of several additional types of cancers (Mersch et al., 2015). CDC20 is involved in
regulation of cell division and responsible for anaphase initiation regulated
by securin degradation. Jiang et al reported that Cdc20 over-expression in
breast tumors when compared to the tissue surrounding the tumors in human
breast cancer patient specimens Jiang et al., 2011. F2(thrombin) in our study was downregulated itself but but has
a positive Z score. It was seen to be connected with overexpression of
chemokines and their ligands which have a significant role in tumor
angiogenesis and cancer metastasis (Kitamura et al., 2015; Bonapace et al.,
2014; Azenshtein et al., 2005). Nierodzik and Karpatkin (2006) has provided ample evidence to support that
thrombin activates tumor cell adhesion to platelets, endothelial cells, and
subendothelial matrix proteins; enhances tumor cell growth; increases tumor
cell seeding and spontaneous metastasis; and stimulates tumor cell
angiogenesis. Further it has also been hypothesized that thrombin plays role in
preservation of dormant tumor cells in individuals, preventing host
eradication. Therefore, it is proposed that tumor malignancy may be regulated
by a procoagulant/anticoagulant axis.

Top upstream regulator identified for benign mammary tumor was
AREG. DNA damage signals caused by radiation and chemo are transmitted by
master regulators like NF-kB to generate a powerful, highly conserved,
cell-non-autonomous and senescence associated secretory phenotype and its
downstream effectors comprise a large spectrum of extracellular proteins,
including AREG, SFRP2, HGF, IL8, MMPs. Together these proteins give rise to a
proangiogenic and proinflammatory microenvironmental niche that promotes
malignant phenotype (Sun et al., 2012; Xu et al., 2016). In our study we
observed its affected downstream molecules as BIRC5, CCNA2, CCNB2, TOP2A, MMP9,
MMP1, CXCR4. AREG mediated activation of EGFR causes an increase in the levels
of stromal fibronectin, which mediates invasion via interaction with integrin
B1 and engagement of PLK1 and BIRC5, genes that are essential for
cancer survival but function independently of EGFR (Kappler et al., 2015). Cytoplasmic
CCNB2 is a potential oncogene and could serve as a potential biomarker
of unfavourable prognosis over short-term follow-up in breast cancer (Shubbar
et al., 2013). The CCNB2 gene is a member of the B-type cyclin family,
including cyclin B1 and B2. It is involved in the G2-M transition in eukaryotes
by activating CDC2 kinase and its inhibition induces cell cycle arrest Draetta
et al., 1989; Wu et al., 2010. In agreement with its crucial role in cell
growth, numerous studies detected overexpression of CCNB2 in human tumors,
including lung, colorectal adenocarcinoma, and pituitary adenomas Hoffman et
al., 2004, de Martino et al., 2009, Stav et al., 2007; Park et al., 2007.
Serum circulating CCNB2 mRNA levels were found to be higher in lung and
digestive tract cancer patients compared to normal controls and were correlated
with cancer stage and metastasis status Mo et al., 2010. CCNA2 is a biomarker
for the prognosis of ER+ breast cancer and monitoring of tamoxifen efficacy Gao
et al., 2014.

Important upstream regulators unique to benign
tumors were AREG, TLR2, TGF1B, HGF, MAP3K1. TLR2 has been reported in
intestinal and breast epithelia oncogenesis. Scheeren et al. (2014) reported
that inhibition of TLR2 or its co-receptor CD14, or its downstream
targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro
and in vivo (Scheeren et al., 2014). Polysaccharide krestin (PSK), a
TLR2 agonist fed to neutransgenic mice significantly inhibited breast cancer
growth by its action on the CD8 (+) T-cell and NK cells but not CD4 (+) T-cells
(Lu et al., 2011). In another study, another TLR2 agonist polysaccharide A
(PSA) was shown to cause inhibition of immune responses by production of IL-10
and regulatory T-cells (Round et al., 2010). Thus, TLR2 stimulation on immune
cells may also have opposing immune effects as in the case of PSA and PSK
(Yousuf et al., 2014).  

Role
of top activated pathways and networks in cancer progression

The top activated pathways in malignant tumors revealed
a unique cancer landscape wherein induction of certain pathways involved
targets associated with cell cycle regulation, cellular proliferation,
apoptotic pathways, cellular stress and injury (e.g., pathways of cell cycle
regulation, estrogen mediated S phase entry, granzyme B signalling and
apoptosis signalling).  The data suggests
genes involved in cell cycle regulation, apoptosis and cell signalling as major
events in the study.

The top scoring network in
mammary tumor was identified around BUB1B which is a protein kinase involved
in metaphase to anaphase transition checkpoint (Li and Murray 1999; Hoyt et
al., 1991). The protein is localized at the kinetochore and plays a role in the
inhibition of the anaphase-promoting complex/cyclosome (APC/C). Aneuploidy and
chromosomal instability (CIN) are common abnormalities in human cancer. BUB1B
is responsible for delaying the onset of anaphase and providing time for proper
chromosome segregation. Alterations of the mitotic spindle checkpoint are
likely to contribute to these phenotypes (Kramer et al., 2002). BUB1B was seen
to be linked with NDC80, an essential protein of kinetochore-associated complex
required for chromosome segregation and spindle checkpoint activity (Tooley and
Stukenbrg et al., 2011). NDC80 complex exists as part of a larger super complex
called KMN network. KMN is brought to centromeres via links to CENP-A and
CENP-C. NDC80 has also been shown to depend on KNL-1 and the CENP/H/I/K complex
for kinetochore recruitment (Desai et al. 2003; Okada et al. 2006; Cheeseman et
al. 2008). KNL-1 is responsible for bringing a separate set of proteins to the
kinetochore. CENP are
centromeric proteins involved in functioning of the kinetochore. We observed a
subnetwork of centromeric proteins CENP- H/C/U/K/A/N in our data. KNL1 and the
CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrate
cells. CENP-H is required for the localisation of CENP-C to the centromere.
However, it may also be involved in the incorporation of newly synthesised
CENP-A into centromere via its interaction with the CENP-A/CENP-HI complex Fakugawa
et al., 2001. It is thought that this protein can bind to itself, as well as
to CENP-A, CENP-B or CENP-C. Multimers of the protein localize constitutively
to the inner kinetochore plate and play an important role in the organization
and function of the active centromere-kinetochore complex Sugata et al., 1999.
Overexpression of CENP H is reported in human breast cancer progression (Laio
et al., 2011), colorectal cancer (Tomonaga et al., 2005), oesophageal cancer
(Guo et al., 2008) oral squamous cell cancer (Shigeishi et al., 2006). Kinetochore protein SPC24 and SPC25 were linked to BUB1B
and overexpressed. KNL1 is likely to associate with the Spc24/Spc25 dimer that
is oriented toward the inner kinetochore. NEK2 was seen linked to BUB1B playing
role in NEK2-mediated phosphorylation of CEP68. This causes CEP68 dissociation
from the centrosome and its degradation at the onset of mitosis. Upregulation
of NEK2 is reported in breast cancer (Hayward et al., 2004).

In benign tumors the top scoring network was
related to cellular assembly and organization, cell cycle, DNA replication,
recombination and repair. The central molecule in this network was vascular
endothelial growth factor (VEGF) , which has been identified as a vascular
permeability factor, angiogenic cytokine, and a survival factor in mammary
tumors. VEGF contributes to mammary tumor growth through increased
neovascularization, as well as autocrine stimulation of growth and inhibition
of apoptosis (Schoeffner et al., 2005). VEGF can act
directly on T lymphocytes and elevated VEGF levels may contribute to the
aberrant MMP-9 secretion by mammary tumor bearing T cells (Owen et al., 2003). VEGF is known
to stimulate IL-18 production, secretion and processing in gastric cancer
cells, and IL-18, in turn, promotes cancer cell migration and proliferation.
Therefore, IL-18 indirectly amplifies cancer cell migration, angiogenesis, and
progression Kim et al., 2007. Besides its activity on tumor invasiveness and
angiogenesis, IL-18 induced expression of a granzyme B inhibitor, protease
inhibitor 9, in gastric cancer cells which decreases their susceptibility to
lymphocyte- mediated cytotoxicity Majima et al., 2009.

Overexpression of Vasohibin 2 (VASH2) was also
seen in our experiment. VASH2 is an angiogenic factor, and has been previously
reported to be a cancer?related gene, with cytoplasmic and karyotypic forms.
VASH2 is a VASH1 homolog, expressed in mononuclear cells and act as an
angiogenesis stimulator in a mouse model of hypoxia-induced subcutaneous
angiogenesis (Kimura et al., 2009). Higher expression of VASH2 induces
expression of growth factors and promotes cell proliferation in breast cancer
(Tu et al., 2014). VASH2 is also involved in the proliferation of hepatic (Xue
et al., 2013) and ovarian (Takahashi et al., 2012; Koyanagi et al., 2013)
cancer. Vasohibin 1 (VASH1) was initially identified as a regulator of negative
feedback in angiogenesis induced by vascular endothelial growth factor (VEGF)
or fibroblast growth factor 2 (FGF2) (Shibuya et al., 2006; Kimura et al.,
2009). Vasohibin-1 (VASH1) is expressed in vascular endothelial cells
stimulated by several angiogenic growth factors and displays autocrine activity
to regulate angiogenesis via a negative feedback mechanism. In our experiment
we observed a higher expression of VASH2 than VASH1.

            Maternal embryonic leucine-zipper kinase
(MELK) encodes a serine/ threonine kinase that is highly conserved
across a variety of mammalian and non-mammalian species. MELK is essential for organogenesis, stem cell
proliferation, and cell cycle regulation. Interestingly, MELK is additionally
involved in the development of numerous human cancers, tumor initiation, and
tumor propagation Nakano et al., 2009; Marie et al., 2008; Kig et al., 2013.
MELK overexpression has been identified in several human cancers: prostate
Kuner et al., 2013, breast Pickard et al., 2009, brain Nakano et al.,
2008, colorectal Gray et al., 2005 and gastric Du et al., 2014. Increased
MELK expression has been detected in particularly aggressive subtypes of breast
cancer such as basal-like breast cancer (BBC), and correlates with poor
prognosis Pickard et a., 2009; Wang et al., 2014. In cancer cells, MELK forms
a protein complex with the transcription factor/oncogene FOXM1, a master
regulator for cell cycle progression Joshi et al., 2013. MELK-regulated
phosphorylation of FOXM1 facilitates FOXM1 transcriptional activity and induces
the expression of various mitotic regulators (e.g. BIRC5, Aurora B, and
CDC25B). MELK may orchestrate the priming event of the complex signaling toward
p53, VEGF, and Wnt/?-catenin in cancers.

In this network we observed upregulation of
ST8SIA4 directly linked to VEGF. ST8SIA4 is involved in sialylation of
proteins. Sialylation is one of the altered glycosylation patterns associated
with cancer progression. Malignant transformations are often associated with a
deregulation of glycosylation processes, and in particular that of terminal
sialylation in breast cancer (Kolbl et a., 2015). Lin et al. (2002) showed that
the cell surface ? 2,6-sialylation contributed to cell–cell and
cell–extracellular matrix adhesion of mammary carcinoma cells, and inhibition
of sialytransferase ST6Gal-I level reduced the metastatic capacity of mammary
carcinoma cells (Lin et al., 2002). Ma et al. (2016) has previously reported
that ST8SIA4 was significantly upregulated in breast tumor tissues as compared
with adjacent tissues. ST8SIA4 is highly correlated with cancer malignancy by
regulating various biological processes (Zhang et al., 2015; Ma et al., 2016).

Overexpression of dual specificity phosphatase (DUSP6)
was seen in our experiment. Some papers report that upregulation of tumor
suppressor genes might promote the malignant phenotype of cancer cells (Hong et
al., 2007). DUSPs are primarily tumor suppressor genes but recent reports show
that depending on the stage of carcinogenesis DUSP1 increases or decreases its
expression (Furukawa et al., 2003; Okudela et al., 2009; Patterson et al.,
2009). This has also been documented for PTEN and IDH1 genes in glioblastoma
cancers (Li et al., 2008; Yan et al., 2009). Overexpression of DUSP6 has been
reported in in estrogen receptor-positive breast cancer cells. Its expression
is strongly upregulated in HER2-positive breast tumors (Lucci et al., 2010).
Moreover, DUSP6 is part of a high-risk signature gene for non-small cell lung
cancer (Chen et al., 2007), and its expression is significantly correlated with
high extracellular signal-regulated kinase (ERK) 1/2 activity in primary human
ovarian cancer cells (Chan et al., 2008). Remarkably, in human glioblastoma
cells DUSP6 gene was induced upon platelet-derived growth factor (PDGF)
stimulation (Tullai et al., 2004; Messina et al., 2011). Moreover, DUSP6 can be
upregulated in specific cancers exhibiting aberrant receptor tyrosine kinase
and Ras/Raf signaling, such as non-small cell lung cancer, potentially as a
negative-feedback regulator of mitogenic signaling (Sato et al., 2006).

Cyclin-dependent kinase (CDK) inhibitor-3 is a
dual-specificity protein tyrosine phosphatase of the CDC14 group (Alonso et
al., 2004). CDKN3 was overexpressed in our experiment. CDKN3 is often
overexpressed in human cancers, with few cases showing CDKN3 gene
amplification. We have found that CDKN3 transcript and protein levels fluctuate
during the cell cycle and peak at mitosis. Since rapidly growing tumors have
more mitotic cells, the high level of CDKN3 in mitotic phase provides the best
plausible explanation for the frequent CDKN3 overexpression in human cancers
(Cress et al., 2017).

Upregulation of PROM1 (CD133) was seen in our
experiment. PROM1 was lined to VEGF directly in this network. PROM1 is linked
to poor prognosis in triple negative breast cancer due to nuclear
mislocalization of PROM1, which normally shows membrane localization and more
sporadically cytoplasmic localization (Cantile et al., 2013). Upregulation of
PROM1 increases the invasive capability and increased expression of proteins
involved in metastasis and drug-resistance of breast tumors (Beaver et al.,
2014; Liu et al., 2013).

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