Hspb1 and Tp53 Mutation and Expression Analysis in Cat Mammary Tumors

1. Background
 The word “cancer” is so fearsome and attention seeking for victims, practitioners and guardiansregardless the type of species afflicted.Mammary adenocarcinoma is the third most common cancer inthe cats (1). Eighty percentofthe total cases are malignant while 10-20% appeared as benign,sooner or later turn into malignant (2). Malignant tumors are equally lethal in animals as they are in humans and several animal cancers e.g. mammary adenocarcinoma in the cat arethe best model forstudying human cancer due tothe resemblance in thecell morphology, histopathology, risk factors and prognosis (3, 4). Mammary tumor is a significant health concern in humans and small animals, so especial emphasis was given to ascertain cancer associatedsequence number variant (SNVs) and gene expression profilingof Hspb1 and Tp53 genes in this neoplasm (5, 6).
 Molecular diagnostic biomarkers are getting much attention now in the field of oncology, but still there are few studies regarding the authentication and usage of these markers as screening tools (7). Disease associated mutations may serve as tumor markers fora particular type of neoplasm. It is one of the major research emphases to diagnose cancer earlier through molecular diagnostics methodologies using single novel signature mutation responsible for the disease outbreak or combination of SNPs or specific haplotype might be helpful for its diagnosis.
 Hspb1gene was characterized in this study which is located on chromosome E3 at 973, 860-975, 895 position, encodes only one transcript of 1411 bp with 3 exons and ciphers 205 amino acids, having 88% and 86% sequence identity with the dog and human counterpart respectively (8). This protein plays its significant role in many processes of tumor development, especially in the cell cycle regulation, immunosurveillance, cell differentiation,and in the apoptotic pathways.High level of this protein wasreported in regression stage of cancer and linked with anti-apoptotic activities (9).
 Tp53 was selected due to being the most variant gene in any type of cancer (10). It is mutated in more than 50% of all malignancies (11). In cats, it is located on E1 chromosome, hasonly one transcript of 1161 bp with 10 exons, and encrypted with 386 amino acid (12). Tp53 protein behaves as a transcription factor, maintains cell growth and genomic integrity (13, 14).
2. Objectives
 The objectives of the current study are to ascertain cancer associatedDNA mutations and expression profiles in Hspb1and Tp53 genes in cancer and disease-free controls. A sensitive and robust, endpoint conventional long-range PCR technique was used to characterize these genes using “Sequencher” software and gene expression profiling through RT-qPCR by TaqMan assay chemistry, which will give us better insight to understand genetic variations and gene expression data simultaneously in cat mammary cancer to improve its clinical diagnosis.

3. Materials and Methods

3.1. Sample Collection
 Six mammary tumor tissues and peripheral bloodof affected Siamese cat including one normal domestic random bred cat were collected throughstandard protocol (Table 1). All neoplastic tissues were excisional biopsies. All tissue masses were storedin -86ºC for DNA/RNA extraction and downstream processes (15).
 
3.2. DNA and Total RNA Extraction
 TaiGen genomic DNA tissue kit (TaiGen Biotechnology Co., Ltd, Neihu Dist., Taipei, Taiwan) was used to extract DNA from the tumorous tissues (16). While genomic DNA from blood was extracted using GF-1 tissue blood combi DNA extraction kit (Vivatis Technologies SDN. BHD. Selangor Darul Ehsan, Malaysia). DNA quantification was done using NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). 50 ng.mL-1  concentration of DNA was used for downstream PCR amplifications.
 Similarly, total  RNA was extracted from cancerous and normal tissues using Thermo Scientific GeneJet RNA purification kit (17) following to the pulverization of the tissuesin the liquid nitrogen. TriZol reagent method was also used to extract total RNA from minute tissues (18). RNA integrity was confirmed by agarose gel electrophoresis and concentration was measured by NanoDrop spectrophotometer.

3.3. Primer and Probes
 Long-range primers were designed from DNA sequence ID ENSFCAT00000026034 and ENSFCAT00000009625 for Hspb1 and Tp53 through the application ofPrimer3 and NetPrimer software (PREMIER Biosoft International, Palo Alto, CA) (19, 20).
Primer express software (Applied Biosystem, USA) was usedto design the primer-probe sequences of theGAPDHgene as an endogenous control for normalization (21). Hspb1and Tp53 primer-probesexpression assays were purchased with FAMflourophore while GAPDH probe was labeled with VIC reporter dye on 5´ end and TAMRA as a quencher on the 3´ (Table 2).

3.4. PCR Amplification
 Long-range PCR was performed using Applied Biosystem thermocycler at 94ºC temperature for 2 min as initial denaturation, then 10 cycles at 94ºC as cyclic denaturation for 10 sec, annealing at 61v for 30 sec and extension temperatures at 68ºC was adopted for 3 and 5 min because the product size were 2303 and 3610 bp in Hspb1 and Tp53 genes respectively. Later on, 30 cycles were run with annealing at 59ºC an dextension was done withan increment of 20 sec per cycle.The final extension was done with at 72ºC for 5 min. Long-range PCR kit was used, which has high-fidelity polymerase with finalconcentration of 1.8 U, PCR 10X enhancer-A withfinal concentration of 1X, PCR additive 5% dimethyl sulfoxide (DMSO) for GC-rich region amplification and 10X reaction buffer with final concentration of 1X (22, 23). 

3.5. Gel Electrophoresis and Data Analysis
 Electrophoresiswith 1.5% agarose gel was conducted for 50 min (Figure 1). Post PCR specificproducts were purified by treating with ExoSAP(24)24] (ExoSAP-IT PCR Product Cleanup, Santa Clara, CA, USA). Sequencing was done with ABI BigDye terminator sequencing Kit (Applied Biosystems, Foster City, CA, USA). “Sequencher” 5.1 software (Gene Codes Corporation, Ann Arbor, MI, USA) was used for sequence analysis (25) (Sequencher® version 5.2 sequence analysis software, Gene Codes Corporation, Ann Arbor, MI USA).

3.6. Reverse Transcription
 Target RNA was reverse transcribed using RevertAid first strand cDNA Synthesis Kit (Thermo Fisher Scientific, Pittsburg, PA, USA) (26). Synthesis of first strand cDNA was performed with oligo (dT) 18 primer and random hexamer primers. 

3.7. RT-qPCR Detection Chemistry and Experimental Design
 TaqMan primer-probe hydrolysis chemistry was adopted by using Applied Biosystem 7500 Real-Time System. Twenty mL reaction volume was used, which contains 1 mL 20X TaqMan assay, 10 mL of 2x TaqMan master mix, 4 mL of cDNA with 5 ng.mL-1 concentration, plus 5 mL of RNase-free water. Then 40 cycles of reaction were run for amplification (Applied Biosystem, USA). All reactions were designed using singleplex two-step qPCR. Both targets (Hspb1 and Tp53) and GAPDH genes were amplified in triplicate in cases and controls and folds change were obtained from Ct values.

3.8. RT-qPCR Data Analysis
 Livak method/DDCt method was used in which fold change expression in cancer (Target) vs. normal samples (Calibrator) and constitutively expressed GAPDH (Reference) genes were calculated by the following formula (27).
        
DCt (Test)              =  Ct (Target)-Ct (Reference)
DCt (Calibrator)     =  Ct (Target)-Ct (Reference)
DDCt                     =  DCt (Test)-DCt (Calibrator)
Fold Change          =  2-DDCt

4. Results

4.1. Hspb1 Mutational Spectrum
 The reference control sequence of random bred cat was usedto align the sequences of our tumour samples (9). Hspb1 gene of the Felis catus has 86% nucleotide identity and 88% protein homology with the human counterpart. No non-synonymous nucleotide alterations were identified in the DNA from five tumorous tissues or in the DNA isolated from blood. Twelve variants were identified in the UTRs, intronic and 5´ flanking regions, but none of them were noticed in cancer cases or in the control. A 4 bp intronic deletion (GTCT) was identified in three cancer samples (CP7, CP16, Cp28), the normal cat has the same sequence of GTCT, but it is absent in the reference sample. Sample (CP16) showed heterozygosity at this position both in somatic tissues and in the blood DNA, which has4 bp deletions in one allele, while the other allele is same as the wild type. Two 5´ UTR and one 3´ UTR mutation showed a gain of heterozygosity in the tumor as compared to the DNA extracted from blood sample within the same individual.
Out of the total ten altered positions excluding UTR and 5´ flanking region, half of the mutation were observed as transversion, while the remaining half appeared as transition mutations (Table 3, Figure 2).

4.2. Tp53 Mutational Landscape
 The overall gain of heterozygosity was observed in the exon 3, 4, and 9 (Table 4). c.105 locus in exon 3 is homozygous (A) in two samples (CP13, CP16) and heterozygous (A/G) in (CP7), but doesn’t change the amino acid. The exon 4 c.465 locus is heterozygous (C/T) in (CP7) while homozygous (C) in another (CP28) sample. Also, itappears to besynonymous. Locus c.859 in exon 7 is homozygous (T) in one sample (CP28) and appeared asa non-sense mutation. Similarly, exon 9 is heterozygous (G/A) at position c.1050 in three cases (CP7, Cp13, CP13A) and proved to be synonymous as well.
 Different polymorphic sites were observed in each of the introns 1, 2, and the 3´flanking region at 202, 278 and 3320 loci, respectively. Intron 7 has two hotspotsat 2167A>A/G and 2217T>T/C. Introns 3, 6, and 8 have three variant positions in each at positions (769, 776, 958), (1756, 1990, 2002), and (2334, 2340, 2415) respectively. Similarly, introns 5 and 9 have different 4 and 7 point mutation correspondingly. Intronic mutations give us clues regardinghow somatic mutations accumulate in a micro-evolutionary process of cancer development. Out of the total 28 polymorphic positions in this gene, 4 positions are transversion, while 24 are transitional changes (Table 4, Figure 2).

4.3. Differential Expression of Tp53
 Two-step singleplex RT-qPCR was conducted on all mammary tumor cancer cases in triplicate and standardized cDNA of 5 ng.mL-1 concentration was used as a template, which was prepared from 100 ng.mL-1 stock RNA. Fold change difference expression values were obtained by using DDCt/comparative Ct method. Calculations were performed using DCt of Tp53 gene in all mammary tumor samples. Similarly, DCt values of GAPDH were also calculated in all cancer samples, while DCt calibrator was calculated (0.61) from the mean Ct target Tp53 subtracted from mean Ct reference/endogenous GAPDH of normal diseased free cat tissues.
    DDCt values were obtained by subtracting DCt target from the DCt calibrator, then this value underwent to thenegative exponential power of 2, which represents the efficiency of the assay. Finally, differential expression values as fold change were obtained mentioned in the Table 3.
 Four mammary tumors were revealed up-regulated forTp53 gene, with the fold change of maximum 344.65, while one sample (CP7) showed down-regulation of this gene (Figure 3).

4.4. Hspb1 Differential Expression
 Similarly, DCt of normal disease-free samples (Calibrator) was calculated (0.54) in Felis catus, which is mean Ct target Hspb1 gene in normal tissues subtracted from the mean Ct of GAPDH from the same normal tissues (Table 4).
     Alike Tp53 up-regulation (in CP13, CP13A, CP16, and CP26), Hspb1 gene is subject ofup-regulation in the same samples, while CP7 sample was foundto become down-regulated same as Tp53 (Figure 4).
5. Discussion
 It is undoubtedly established that accumulation of mutations leads to cancer or cell death. Hspb1 as a diagnostic marker are not so much informative, but they are expedient indicators for carcinogenesis in some tissues and predict the differentiation and aggressiveness of few cancers (28). This gene has been widely studied in human cancers (29), it has hotspot motif at functional promoter rs2868371 and G1271C (30). Similarly in veterinary species, Austrian feline solid carcinoma of mammary glands, where Arg CGG-TGG Tyr variant was found in exon 8 of Tp53 (31). The current study did not report this variant probably due to breed or geographical population differences. But it has been found that Tp53 exon 3, 4, 7 and 9 are more variable as compared to other exons and codon 35, 155, 287 and 350 were found mutant. Codons 35, 155, and 350 are synonymous while thecodon 287 terminates the peptide chain and turned into the stop codon.
 Tp53 gene was characterized in FVAS where 8 SNPs along with (T) insertions were found in exon 5, 6, 7 and 8 (32). Codon 163 and positions 14,246, 247, and 259 of intron 7 were also altered (33). The change inthe c.105 locus of the Tp53 in samples CP7 (c.105G>A/G), CP13 (c.105G>A) and CP16 (c.105G>A) were observed (Table 2). The same locus was reported polymorphic in human pancreatic cancer (34), hepatocarcinoma (35), breast and ovarian cancers (36).
 Locus c.465C>T of the Tp53 was found altered in CP7 (c.465T>T/C) and CP28 (c.465T>C) samples (Table 2). Same locuswerereported to be altered in eight different studies in human breast cancer (37, 38), ovarian cancer (39), hepatocarcinoma (40), colorectal cancer (41), endometrial tumor (42), sinonasal cancer (43), esophageal adenocarcinoma (44), while one study has shownc.465C>A change, which was on esophageal SCC in Chinese population (45). This locus appeared as synonymous in all these studies.
 Locus c.859 was also found very informative in human studies, as we observed here in cat mammary tumor of CP28 sample, this transversion of (c.859G>T) turned into stop codon in this sample which is a transitional change of (c.859G>A) in DNA binding site of coding strand in five human studies of bladder cancer (46), hepatocellular carcinoma (47), skin SCC (48), aerodigestive tract (49), gastric cancer (50), and changed glutamic acid to lysine (p.E287K) inthe protein. Another transversion change of c.859G->T was also noticed in thefive different studies on this locus including Burkitt Lymphoma and chronic lymphocytic leukemia (51), non-small-cell lung cancer (52), bladder carcinoma (53), thyroid carcinoma (54), esophageal carcinomas (55) in whichglutamic acid was changed to stop a codon (p.E287X) in the protein as well as in the presentstudy in this cat mammary tumor (CP28) (Table 2).
 The c.1050 position of the Tp53 in cats mammary tumor was found altered in three cancer samples (CP7, CP13, CP13A), in which c.1050G>G/A change was observed (Table 2), which appeared as synonymous. In one of the human aristolochic acid-associated urothelial cancer in Taiwan population, this locus was also found to be altered at c.1050C->G and synonymous in nature.This point mutation was transversion in the DNA coding strand, which encodes an amino acid in alpha helix structure of this protein (56). Our studied mammary cases have shown alterations in exon 3, 4, 7, and 9 of Tp53 gene. A 4 bp deletion was found in the intron 2 of the Hspb1 gene in the three cat mammary tumors (CP7, CP16, CP28), which is homozygous in two (CP7, CP28), and heterozygous in one neoplastic tissue, as well asthe blood of one animal (CP16, CP16b) samples.

5.1. Comparison of Hspb1 Polymorphism
 Cross-tissue (germ-line vs somatic) mutational comparison of the cat’s Hspb1 gene was re-evaluated and revealed that the mostly altered loci are same in neoplastic tissues and blood of the same animal CP13 and CP16, especially in the exonic regions, but few of the intronic positions show heterozygosity. Similarly, (-)166 locus in 5´UTR of exon 1 in two cases: CP13 and CP16 acquired the same heterozygous (T/A) mutation in tumorous tissues while blood DNA are homozygous (A) at this locus. In sample CP13at 773 position of 3´UTR of Hspb1 gene found heterozygous (A/T) in cancerous tissues, while homozygous (A) in the blood (Table 1). Other polymorphic loci were observed to be the same in both tissues. Similarly, 3´UTR locus 1868 in CP13 tumorous tissue is also different from the blood. It is homozygous (A) in blood, while heterozygous (A/T) in neoplastic tissues (Table 2).

5.2. Comparison of Tp53 Polymorphism
 Cross-tissue mutation comparison (germ-line versus somatic) was also conducted for Tp53. Comparison of sample ID CP13 and CP16 was conducted which revealed that exonic regions are the same between the two tissue types while five loci in the intronic region in CP13 are different in this cross-examination of polymorphic sites. Few loci in blood are different e.g. gene position 1555 in intron 5 is heterozygous (G/C) in blood instead of homozygous (G) in neoplastic tissue, position 2167 in intron 7 is homozygous (A) in blood instead of heterozygous (A/G) in cancerous tissue. Similarly, two positions 2521 and2854 in intron 9 are homozygous (G) and (A) instead of heterozygous (G/A) in neoplastic tissues of the sample CP13. CP16 sample was found heterozygous (C/T) instead of homozygous (C) in cancerous tissues in intron 3 at position 769 (Table 2).
 Regarding the Hspb1 and Tp53 expression in relation to different mutations, up-regulation of Tp53 was observed in 4 mammary tumors (CP13, CP13A, CP16, and CP28). Two tumors samples (CP13, CP16) showed fold change of 2.62 and 54.19 respectively, while two of the tumor samples (CP13A, CP28) showed almost same up-regulation of 344.57 and 344.65, respectively. Significant up-regulation of Tp53 expression, as compared to the calibrator, is much more informative in differential diagnosis as compared to those markers which haveslightly higher over-expression (Table 3, Figure 3).
 Tp53 up and down regulated samples are significantly different in their mutational landscape, as up-regulated samples (CP13, CP16) have 105G>A common mutation in exon 3. Likewise, (CP13, CP13A) have common mutation of 1050G>G/A in exon 9, while up-regulated sample (CP28) was quite different from their counterparts, which has two homozygous variants of 465T>C in exon 4and 859G>T in exon 7. Different mutations in these four samples which are different from the down-regulated sample are 202C in intron 1, 278G in intron 2, 776C in intron 3, 1474T in intron 5 in (CP13, CP13A, and CP16), while 202G, 278C, 776T, 1474C in (CP28).     
 Down-regulated sample (CP7) of Tp53 gene were found heterozygous at 105G>G/A in exon 3, 465T>T/C in exon 4 and 1050G>G/A in exon 9 loci, which are homozygous (A), (T) and (G) in up-regulated samples of the mammary tumors respectively. These loci 202C/G, 769C, 278G, 1474T/C, 1555C, 2737G/A, 2941G/A of (CP7) are different from the above mentioned up-regulated samples (Table 3).Foursamples (CP13, CP13A, CP16, and CP28) showed up-regulation of theHspb1 out of the total five tumors (Table 4, Figure 4). Minimum up-regulation of 100.22 fold change was observed in one sample (CP28). One of the tumor sample CP13A showed almost same expression of Hspb1 and Tp53 in the range of 327.65 and 344.57 respectively. The up-regulation trend of Hspb1 gene expression can be correlated with common heterozygous mutations of 166T>T/A, 286A>A/G, 305T>T/C, and 388C>C/T in three up-regulated samples (CP13, CP13A, and CP16) in exon 1, while (CP28) sample has up-regulation of Hspb1 gene with all homozygous changes on the same loci 166T>A, 286A>G, 305T>C, and 388C>T.
 One of the worth mentioning change was 1514-1517del4 that has been observed in all four up-regulated samples in intron 2 of Hspb1. Similarly, 1326T>T/C change was observed in intron 1 and 1868A>A/T in 3´UTR in two up-regulated samples (CP13, CP13A) while the third up-regulated sample (CP16) was found heterozygous on 1514-1517 locus with additional change of 1490C>C/G in intron 2. The fourth up-regulated sample of (CP28) showed 1514-1517del4 change alongwith an additional change of 1868A>A/T in 3´UTR. One mammary tumor sample (CP7) showed down-regulation of Hspb1 as compared to the calibrator, which showed fold change of 0.42.Down-regulation might be associated with homozygous locus of 286A>G, 305T>C and 338C>T. Down-regulated sample has homozygous variant on the same three loci as compared to the heterozygous variants in up-regulated samples. These changes are similar to (CP28) but with the difference inone locus of 166T>A. Intronic regions are also found altered in this single down-regulated sample (CP7), in which 1514-1517Del4 and 1490C>G changes were found in the intron 2.   
   
6. Conclusions
 Tp53 was found to be more polymorphic than Hspb1. Exon 3, 4 and 9 have one synonymous mutant site in each, while one mutant in exon 7 was appeared as non-sense. Introns 1, 2 and 9 were found polymorphic with 1, 1, and 7 variants respectively. Introns 3, 6 and 8 have three mutant loci while intron 5 was observed with four mutant loci. Intron 6 has an insertion of 1 bp at the position 1990 in sample CP28. Similarly, exon 1 in Hspb1 has six polymorphic sites with one synonymous mutation, while remaining five are located in the upstream region. Exon 3 was also observed mutated at a single at genomic position of 1326 in its intronic region. Regarding the gene expression, overall up-regulation of the both genes was observed in this cat neoplasm as compared to normal.

Acknowledgements
 Authors are thankful to HEC-Pakistan, management of University of Missouri-Columbia, USA, Institute of Biochemistry and Biotechnology and Pet center University of Veterinary and Animal Sciences, Lahore-Pakistan.