The top upregulated genes amongmalignant and benign tumors in our study were found to have a significant rolein cancer progression and tumor invasiveness, as evidenced by human cancerstudies.

Top five most upregulated genes in malignanttumors were COL11A1, SFRP2, LCN2, COL2A1 & H19. COL11A1gene encodes one of the two alpha chains of type XI collagen, a minor fibrillarcollagenwhich had been implicated in tumorprogression in humans. Overexpression of COL11A1 has been reported inmesenchymal 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 etal., 2008).  Overexpression is correlatedwith poor clinical outcome in human ovarian cancer and advanced stages ofcancers.

Wu et al., reported that COL11A1 may promote tumor aggressiveness viathe TGF-?1 and MMP3 axis (Wu et al., 2014). Halstead et al. (2008)reported expression of COL11A1 in epithelial cells, stroma, and vessels ofnormal and cancerous breast tissue (Halstead et al., 2008).             Secretedfrizzled-related proteins (SFRPs), constitute a family of 5 members (SFRP 1–5)that modulate Wnt signaling. These proteins are presumed extracellular Wntinhibitors.

 Overexpression of secretedfrizzled related protein 2 (SFRP2) incanine mammary gland tumors has been shown to induce cancerous transformation in normal mammary epithelialcells (Leeet al. 2004). SFRP2 is localized in the extracellular matrix of mammary glandtumors. The anti-apoptotic function of SFRP2 is mediated through activation of NF-?Bor Janus kinase (JNK) suppression (Lee et al., 2006). Further, in vitro, as well as, in vivo oncogenic potential of SFRP2 has been demonstrated inrenal cancer.

Recently,SFRP2 has been related to poor prognosis along with genes associatedwith epithelial-to-mesenchymal transition (Vincent et al., 2017). Overexpression of SFRP2 was seen in both malignantand benign mammary tumors in our experiment.            Lipocalin (LCN2), another top upregultaed gene amongmalignant tumors in our data set, has been shown to be associated with estrogenreceptor (ER)-negative breast tumors in humans (Gruvberger et al.

, 2001). Thegene is significantly increased in the luminal epithelial cells compared withmyoepithelial cells (Jones et al., 2004), an important finding because themajority of breast carcinomas are thought to arise from the luminal epithelial cells(Rakha et al.

, 2008). LCN2promotes breast cancer progression by inducing epithelial to mesenchymaltransition (EMT) through the ER?/Slug axis (Yang et al., 2009). LCN2 also playsan important role in promoting cell migration and invasion of prostate cancerby inducing epithelial to mesenchymal transition through the ERK/SLUG axis(Ding et al., 2015). Recentlyit has been shown to be associated with tumor invasiveness of human cervicalcancer (Chung et al.

,2016)Ganpathi etal., (2016) revealed that high expression of collagen type II, alpha 1 (COL2A1)was associated with delayed time to recurrence in high grade serious ovariancancers. COL2A1 gene has been reported to undergosomatic alterations in cases of  chondrosarcoma and enchondroma cases (Totokiet 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 reportedH19, to have key regulatory functions in tumor development and progression (Brannan etal., 1990).

Dugimont(1995) reported expression of H19 gene in both epithelial and stromalcomponents of human invasive adenocarcinomas (Dugimont et al., 1995). H19levels have also been reported to be remarkably increased in bladder cancertissues. H19 promotes bladder cancer cell migration in vitro and invivo. H19 is associated with enhancer of EZH2, and this association resultsin Wnt/?-catenin activation and subsequent downregulation of E-cadherin.

H19 hasbeen reported to enhance bladdercancer metastasis by associating with EZH2 and inhibiting E-cad expression (Luoet al., 2013). Recently biological and clinical relevance of H19 in Colorectalcancer patients have been thoroughly covered by   Schwarzenbach, 2016The most upregulated genes among benign mammarytumors in our experiment were MMP3, MMP1, AREG, PTHLH and SFRP2. Matrixmetalloproteinases(MMPs) play role in cancer progression by degradingextracellular matrix and basement membrane and are the main proteolytic enzymesinvolved in cancer invasion and metastasis (Stamenkovic, 2000). MMP3 and MMP1have a synergistic effect on breast cancer carcinogenesis (Padala et al., 2017).MMP1 is the most widely expressed collagenase and plays role in degradation ofcollagen I, II and III. MMP-3 has a high proteolytic efficiency and activates anumber of proMMPs (Visse et al.

, 2003; Lui et al., 2012). Amphiregulin (AREG) is one of the many ligands for epithelialgrowth factor receptor (Luetteke et al., 1999).

It plays a central role inmammary gland development and branching morphogenesis in organs and isexpressed both in healthy and cancerous tissues (Plowman et al., 1990, Ciarloniet al., 2007, Kariagina et al., 2010, McBryan et al., 2008). Various studieshave highlighted the functional role of AREG in several aspects oftumorigenesis, 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 ofAREG has already been described in the most common human epithelialmalignancies, 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, Turringet al.

, 1998). Furthermore, AREG is also present in the tumor microenvironment (TME)and contributes to therapeutic resistance toseveral cancer treatments (Ishikawa et al., 2005, Eckstein et al., 2008, Xu etal 2016). Parathyroid hormone like hormone(PTHLH) has previously been reported to be produced by tumor cells in the bonemicroenvironment and is implicated in osteoclastic activity and contribute toosteolytic bone metastasis (Broaduset al., 1988).

Ghoussaini and colleagues combined several datasets for agenome wide analysis and identified PTHLH as loci for susceptibility for breastcancer (Ghoussaini et al., 2012). Researchers suggest that PTHLP powerfullypromotes tumor formation in breast cancer (Li et al.

, 2011). Impact analysis of major upstream regulators In our study we identified numerous dysregulatedupstream regulators in both malignant and benign tumors of mammary gland, whichaffected expression patterns of various other genes related with tumorogeneis. Thetop upstream regulators in malignant mammary tumors were secretedphosphoprotein (SPP1) and coagulation factor II (F2). Pathway analysisrevealed that secreted phosphoprotein (SPP1) overexpression affected the expression levels of breast cancer type1 susceptibility protein(BRCA1) & Cell-division cycle protein 20 (CDC20). BRCA1 is a transcription regulator implicated inbreast cancer pathogenesis and havebeen associated with increased risks of several additional types of cancers (Mersch et al., 2015). CDC20 is involved inregulation of cell division and responsible for anaphase initiation regulatedby securin degradation.

Jiang et al reported that Cdc20 over-expression inbreast tumors when compared to the tissue surrounding the tumors in humanbreast cancer patient specimens Jiang et al., 2011. F2(thrombin) in our study was downregulated itself but but hasa positive Z score. It was seen to be connected with overexpression ofchemokines and their ligands which have a significant role in tumorangiogenesis 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 thatthrombin activates tumor cell adhesion to platelets, endothelial cells, andsubendothelial matrix proteins; enhances tumor cell growth; increases tumorcell seeding and spontaneous metastasis; and stimulates tumor cellangiogenesis. Further it has also been hypothesized that thrombin plays role inpreservation of dormant tumor cells in individuals, preventing hosteradication. Therefore, it is proposed that tumor malignancy may be regulatedby a procoagulant/anticoagulant axis.Top upstream regulator identified for benign mammary tumor wasAREG. DNA damage signals caused by radiation and chemo are transmitted bymaster regulators like NF-kB to generate a powerful, highly conserved,cell-non-autonomous and senescence associated secretory phenotype and itsdownstream effectors comprise a large spectrum of extracellular proteins,including AREG, SFRP2, HGF, IL8, MMPs.

Together these proteins give rise to aproangiogenic and proinflammatory microenvironmental niche that promotesmalignant phenotype (Sun et al., 2012; Xu et al., 2016). In our study weobserved its affected downstream molecules as BIRC5, CCNA2, CCNB2, TOP2A, MMP9,MMP1, CXCR4. AREG mediated activation of EGFR causes an increase in the levelsof stromal fibronectin, which mediates invasion via interaction with integrinB1 and engagement of PLK1 and BIRC5, genes that are essential forcancer survival but function independently of EGFR (Kappler et al.

, 2015). CytoplasmicCCNB2 is a potential oncogene and could serve as a potential biomarkerof unfavourable prognosis over short-term follow-up in breast cancer (Shubbaret 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 eukaryotesby activating CDC2 kinase and its inhibition induces cell cycle arrest Draettaet al., 1989; Wu et al., 2010. In agreement with its crucial role in cellgrowth, numerous studies detected overexpression of CCNB2 in human tumors,including lung, colorectal adenocarcinoma, and pituitary adenomas Hoffman etal., 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 anddigestive tract cancer patients compared to normal controls and were correlatedwith cancer stage and metastasis status Mo et al., 2010. CCNA2 is a biomarkerfor the prognosis of ER+ breast cancer and monitoring of tamoxifen efficacy Gaoet al., 2014. Important upstream regulators unique to benigntumors were AREG, TLR2, TGF1B, HGF, MAP3K1.

TLR2 has been reported inintestinal and breast epithelia oncogenesis. Scheeren et al. (2014) reportedthat inhibition of TLR2 or its co-receptor CD14, or its downstreamtargets MYD88 and IRAK1 inhibits growth of human breast cancers in vitroand in vivo (Scheeren et al., 2014). Polysaccharide krestin (PSK), aTLR2 agonist fed to neutransgenic mice significantly inhibited breast cancergrowth 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-10and regulatory T-cells (Round et al., 2010).

Thus, TLR2 stimulation on immunecells may also have opposing immune effects as in the case of PSA and PSK(Yousuf et al., 2014).  Roleof top activated pathways and networks in cancer progressionThe top activated pathways in malignant tumors revealeda unique cancer landscape wherein induction of certain pathways involvedtargets associated with cell cycle regulation, cellular proliferation,apoptotic pathways, cellular stress and injury (e.

g., pathways of cell cycleregulation, estrogen mediated S phase entry, granzyme B signalling andapoptosis signalling).  The data suggestsgenes involved in cell cycle regulation, apoptosis and cell signalling as majorevents in the study. The top scoring network inmammary tumor was identified around BUB1B which is a protein kinase involvedin metaphase to anaphase transition checkpoint (Li and Murray 1999; Hoyt etal., 1991). The protein is localized at the kinetochore and plays a role in theinhibition of the anaphase-promoting complex/cyclosome (APC/C).

Aneuploidy andchromosomal instability (CIN) are common abnormalities in human cancer. BUB1Bis responsible for delaying the onset of anaphase and providing time for properchromosome segregation. Alterations of the mitotic spindle checkpoint arelikely to contribute to these phenotypes (Kramer et al.

, 2002). BUB1B was seento be linked with NDC80, an essential protein of kinetochore-associated complexrequired for chromosome segregation and spindle checkpoint activity (Tooley andStukenbrg et al., 2011). NDC80 complex exists as part of a larger super complexcalled KMN network. KMN is brought to centromeres via links to CENP-A andCENP-C. NDC80 has also been shown to depend on KNL-1 and the CENP/H/I/K complexfor kinetochore recruitment (Desai et al. 2003; Okada et al.

2006; Cheeseman etal. 2008). KNL-1 is responsible for bringing a separate set of proteins to thekinetochore. CENP arecentromeric proteins involved in functioning of the kinetochore. We observed asubnetwork of centromeric proteins CENP- H/C/U/K/A/N in our data.

KNL1 and theCENP-H/I/K complex coordinately direct kinetochore assembly in vertebratecells. CENP-H is required for the localisation of CENP-C to the centromere.However, it may also be involved in the incorporation of newly synthesisedCENP-A into centromere via its interaction with the CENP-A/CENP-HI complex Fakugawaet al.

, 2001. It is thought that this protein can bind to itself, as well asto CENP-A, CENP-B or CENP-C. Multimers of the protein localize constitutivelyto the inner kinetochore plate and play an important role in the organizationand function of the active centromere-kinetochore complex Sugata et al., 1999.Overexpression of CENP H is reported in human breast cancer progression (Laioet 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 BUB1Band overexpressed. KNL1 is likely to associate with the Spc24/Spc25 dimer thatis oriented toward the inner kinetochore. NEK2 was seen linked to BUB1B playingrole in NEK2-mediated phosphorylation of CEP68. This causes CEP68 dissociationfrom the centrosome and its degradation at the onset of mitosis. Upregulationof NEK2 is reported in breast cancer (Hayward et al.

, 2004). In benign tumors the top scoring network wasrelated to cellular assembly and organization, cell cycle, DNA replication,recombination and repair. The central molecule in this network was vascularendothelial growth factor (VEGF) , which has been identified as a vascularpermeability factor, angiogenic cytokine, and a survival factor in mammarytumors. VEGF contributes to mammary tumor growth through increasedneovascularization, as well as autocrine stimulation of growth and inhibitionof apoptosis (Schoeffner et al., 2005). VEGF can actdirectly on T lymphocytes and elevated VEGF levels may contribute to theaberrant MMP-9 secretion by mammary tumor bearing T cells (Owen et al., 2003).

VEGF is knownto stimulate IL-18 production, secretion and processing in gastric cancercells, and IL-18, in turn, promotes cancer cell migration and proliferation.Therefore, IL-18 indirectly amplifies cancer cell migration, angiogenesis, andprogression Kim et al., 2007. Besides its activity on tumor invasiveness andangiogenesis, IL-18 induced expression of a granzyme B inhibitor, proteaseinhibitor 9, in gastric cancer cells which decreases their susceptibility tolymphocyte- mediated cytotoxicity Majima et al.

, 2009. Overexpression of Vasohibin 2 (VASH2) was alsoseen in our experiment. VASH2 is an angiogenic factor, and has been previouslyreported to be a cancer?related gene, with cytoplasmic and karyotypic forms.VASH2 is a VASH1 homolog, expressed in mononuclear cells and act as anangiogenesis stimulator in a mouse model of hypoxia-induced subcutaneousangiogenesis (Kimura et al., 2009).

Higher expression of VASH2 inducesexpression of growth factors and promotes cell proliferation in breast cancer(Tu et al., 2014). VASH2 is also involved in the proliferation of hepatic (Xueet al., 2013) and ovarian (Takahashi et al., 2012; Koyanagi et al.

, 2013)cancer. Vasohibin 1 (VASH1) was initially identified as a regulator of negativefeedback 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 cellsstimulated by several angiogenic growth factors and displays autocrine activityto regulate angiogenesis via a negative feedback mechanism. In our experimentwe observed a higher expression of VASH2 than VASH1.             Maternal embryonic leucine-zipper kinase(MELK) encodes a serine/ threonine kinase that is highly conservedacross a variety of mammalian and non-mammalian species.

MELK is essential for organogenesis, stem cellproliferation, and cell cycle regulation. Interestingly, MELK is additionallyinvolved in the development of numerous human cancers, tumor initiation, andtumor propagation Nakano et al., 2009; Marie et al., 2008; Kig et al.

, 2013.MELK overexpression has been identified in several human cancers: prostateKuner et al., 2013, breast Pickard et al., 2009, brain Nakano et al.

,2008, colorectal Gray et al., 2005 and gastric Du et al., 2014. IncreasedMELK expression has been detected in particularly aggressive subtypes of breastcancer such as basal-like breast cancer (BBC), and correlates with poorprognosis Pickard et a.

, 2009; Wang et al., 2014. In cancer cells, MELK formsa protein complex with the transcription factor/oncogene FOXM1, a masterregulator for cell cycle progression Joshi et al., 2013. MELK-regulatedphosphorylation of FOXM1 facilitates FOXM1 transcriptional activity and inducesthe expression of various mitotic regulators (e.g.

BIRC5, Aurora B, andCDC25B). MELK may orchestrate the priming event of the complex signaling towardp53, VEGF, and Wnt/?-catenin in cancers.In this network we observed upregulation ofST8SIA4 directly linked to VEGF. ST8SIA4 is involved in sialylation ofproteins. Sialylation is one of the altered glycosylation patterns associatedwith cancer progression.

Malignant transformations are often associated with aderegulation of glycosylation processes, and in particular that of terminalsialylation in breast cancer (Kolbl et a., 2015). Lin et al. (2002) showed thatthe cell surface ? 2,6-sialylation contributed to cell–cell andcell–extracellular matrix adhesion of mammary carcinoma cells, and inhibitionof sialytransferase ST6Gal-I level reduced the metastatic capacity of mammarycarcinoma cells (Lin et al.

, 2002). Ma et al. (2016) has previously reportedthat ST8SIA4 was significantly upregulated in breast tumor tissues as comparedwith adjacent tissues.

ST8SIA4 is highly correlated with cancer malignancy byregulating 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 tumorsuppressor genes might promote the malignant phenotype of cancer cells (Hong etal.

, 2007). DUSPs are primarily tumor suppressor genes but recent reports showthat depending on the stage of carcinogenesis DUSP1 increases or decreases itsexpression (Furukawa et al., 2003; Okudela et al., 2009; Patterson et al.,2009). This has also been documented for PTEN and IDH1 genes in glioblastomacancers (Li et al.

, 2008; Yan et al., 2009). Overexpression of DUSP6 has beenreported in in estrogen receptor-positive breast cancer cells.

Its expressionis 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 lungcancer (Chen et al., 2007), and its expression is significantly correlated withhigh extracellular signal-regulated kinase (ERK) 1/2 activity in primary humanovarian cancer cells (Chan et al., 2008).

Remarkably, in human glioblastomacells DUSP6 gene was induced upon platelet-derived growth factor (PDGF)stimulation (Tullai et al., 2004; Messina et al., 2011). Moreover, DUSP6 can beupregulated in specific cancers exhibiting aberrant receptor tyrosine kinaseand Ras/Raf signaling, such as non-small cell lung cancer, potentially as anegative-feedback regulator of mitogenic signaling (Sato et al.

, 2006).Cyclin-dependent kinase (CDK) inhibitor-3 is adual-specificity protein tyrosine phosphatase of the CDC14 group (Alonso etal., 2004).

CDKN3 was overexpressed in our experiment. CDKN3 is oftenoverexpressed in human cancers, with few cases showing CDKN3 geneamplification. We have found that CDKN3 transcript and protein levels fluctuateduring the cell cycle and peak at mitosis. Since rapidly growing tumors havemore mitotic cells, the high level of CDKN3 in mitotic phase provides the bestplausible explanation for the frequent CDKN3 overexpression in human cancers(Cress et al., 2017). Upregulation of PROM1 (CD133) was seen in ourexperiment. PROM1 was lined to VEGF directly in this network. PROM1 is linkedto poor prognosis in triple negative breast cancer due to nuclearmislocalization of PROM1, which normally shows membrane localization and moresporadically cytoplasmic localization (Cantile et al.

, 2013). Upregulation ofPROM1 increases the invasive capability and increased expression of proteinsinvolved in metastasis and drug-resistance of breast tumors (Beaver et al.,2014; Liu et al., 2013).


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