Gene Expression Profiling of Triple-Negative Breast Tumors with Different Expression of Secreted Protein Acidic And Cysteine Rich (SPARC)

Figures & data

Table 1.  Clinicopathological features of triple-negative breast cancer patients.

Variable	Characteristic	n (%)
Age (years)	Median 48 (range 24–77)
TNM stage	Early	20 (39.2)
	Late	20 (39.2)
	Missing	11 (21.6)
Tumor size	pT1 + pT2	41 (80.4)
	pT3 + pT4	10 (19.6)
Lymph node status	pN0	29 (56.9)
	pN^+	21 (41.2)
	Missing	01 (2.0)
Histological grade	I + II	07 (13.7)
	III	44 (86.3)
Nuclear grade	II	03 (5.9)
	III	47 (92.2)
	Missing	01 (2.0)

Tumor-node-metastasis (TNM) stage: Patients’ stage determined according to the 5th Edition of the Union for International Cancer Control (UICC) TNM classification of malignant tumors.

Table 2.  50 top genes differentially expressed identified in transcriptome analysis: SPARC positive versus SPARC negative – positively regulated.

Gene symbol	Description	Fold change	Associated with cancer
SOHLH2	Spermatogenesis and oogenesis-specific basic helix-loop-helix 2	24.20	Yes^†
KLHL13	Kelch-like family member 13	22.09	No
HPGD	Hydroxyprostaglandin dehydrogenase 15-(NAD)	20.01	Yes^‡
GPR158	G-protein-coupled receptor 158	18.73	Yes
FABP7	Fatty acid-binding protein 7, brain	18.30	Yes^‡
CTAG1A	Cancer/testis antigen 1A	18.09	Yes
ZNF556	Zinc finger protein 556	17.34	No
SLC26A4	Solute carrier family 26 (anion exchanger), member 4	15.66	Yes^†
MAGEA1	Melanoma antigen family A, 1	14.49	Yes^‡
UGT2B11	UDP glucuronosyltransferase 2 family, polypeptide B11	14.13	Yes^†
SLAIN1	SLAIN motif family, member 1	13.44	Yes
C6orf105	Androgen-dependent TFPI-regulating protein	13.05	No
GSTM2P1	Glutathione S-transferase mu 2 (muscle) pseudogene 1	13.04	No
HS3ST4	Heparan sulfate (glucosamine) 3-O-sulfotransferase 4	12.58	No
CYP26A1	Cytochrome P450, family 26, subfamily A, polypeptide 1	12.56	Yes^†
TOX3	TOX high-mobility group box family member 3	12.52	Yes^†
FBN2	Fibrillin 2	11.53	Yes^†
FSIP1	Fibrous sheath-interacting protein 1	11.29	Yes^†
CDH1	Cadherin 1, type 1, E-cadherin (epithelial)	10.64	Yes^‡
ZNF385B	Zinc finger protein 385B	10.52	Yes^†
TDRD9	Tudor domain containing 9	9.71	No
CHRDL2	Chordin-like 2	9.67	Yes^†
CELF4	CUGBP, Elav-like family member 4	9.10	Yes
TNFRSF17	Tumor necrosis factor receptor superfamily, member 17	8.63	Yes^†
DDC	Dopa decarboxylase (aromatic L-amino acid decarboxylase)	8.53	Yes
KIRREL3	Kin of IRRE like 3 (Drosophila)	8.31	Yes
FBXW10	F-box and WD repeat domain containing 10	8.20	Yes
GLYATL2	Glycine-N-acyltransferase-like 2	8.13	Yes
TRIM36	Tripartite motif containing 36	7.99	Yes^†
MSMB	Microseminoprotein, β-	7.97	Yes^†
ZNF204P	Zinc finger protein 204, pseudogene	7.63	No
SEMA3D	Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3D	7.47	Yes
TSPAN8	Tetraspanin 8	7.41	Yes
MUC20	Mucin 20, cell surface associated	7.37	Yes
APOD	Apolipoprotein D	7.33	Yes^†
C8orf46	Chromosome 8 open-reading frame 46	7.31	No
ATP1A2	ATPase, Na^+/K^+ transporting, α 2 polypeptide	7.26	Yes^†
MYOM2	Myomesin 2	7.02	Yes^†
FAM65B	Family with sequence similarity 65, member B	6.78	Yes
CLDN1	Claudin 1	6.69	Yes^‡
RIMS2	Regulating synaptic membrane exocytosis 2	6.67	Yes
SC5DL	Sterol-C5-desaturase	6.56	No
PPM1H	Protein phosphatase, Mg^2+/Mn^2 ^+ dependent, 1H	6.51	Yes^†
MAGEC1	Melanoma antigen family C, 1	6.26	Yes^†
MSLN	Mesothelin	6.26	Yes^‡
EAF2	ELL-associated factor 2	6.14	Yes
AMTN	Amelotin	6.14	Yes
IGLL5	Immunoglobulin lambda-like polypeptide 5	6.07	Yes^†
FAM70A	Transmembrane protein 255A	5.97	No
EGF	Epidermal growth factor	5.87	Yes^‡

^†Associated with breast cancer.

^‡Associated with TNBC.

TNBC: Triple-negative breast cancer.

Figure 1.  Heat map depicting the relative fold change in expression of differentially expressed genes in the comparison of TNBC/SPARC+ vs TNBC/SPARC-.

The hierarchical cluster shows the fold change inexpression of the 50 TOP upregulated genes and 50 TOP downregulated genes, using the cut off foldchange ≥ 3. Columns in heat map correspond to the samples categorized as TNBC with SPARC positive expression and good prognosis (in grey) and TNBC with SPARC negative expression and poor prognosis (in black). Transcript enrichment is encoded in the heat map in green when showing downregulation; or red when showing upregulation in the comparison of TNBC/SPARC+ vs TNBC/SPARC.

TNBC: Triple negative breast cancer; TOP: Gene expression profiling of TNBC with differences in SPARC expression.

Figure 2.  Molecular interaction network of the downregulated genes in the comparison of TNBC/SPARC+ vs. TNBC/SPARC-.

Genes or gene products are represented as nodes, and the biological relationship betweentwo nodes is represented as an edge. The intensity of the node color indicates the degree of upregulation (red) or downregulation (green). Edges are displayed with various labels that describe the nature of the relationship between the nodes: — binding only, → acts on, Θ auto-regulation.

Table 3.  50 top genes differentially expressed identified in transcriptome analysis: SPARC positive versus SPARC negative – negatively regulated.

Gene symbol	Description	Fold change	Associated with cancer
KRT1	Keratin 1	-57.75	Yes^‡
CAPN8	Calpain 8	-33.00	No
IVL	Involucrin	-32.16	Yes^†
SPRR3	Small proline-rich protein 3	-31.54	Yes^†
SCGB2A2	Secretoglobin, family 2A, member 2	-30.474	Yes^†
SCGB2A1	Secretoglobin, family 2A, member 1	-25.594	Yes^†
LHX1	LIM homeobox 1	-22.51	Yes
CEACAM7	Carcinoembryonic antigen-related cell adhesion molecule 7	-21.611	Yes^†
DDTL	D-dopachrome tautomerase-like	-21.601	No
MesP1	Mesoderm posterior basic helix-loop-helix transcription factor 1	-21.20	No
CEACAM6	Carcinoembryonic antigen-related cell adhesion molecule 6 (nonspecific cross-reacting antigen)	-19.95	Yes^‡
FAM25A	Family with sequence similarity 25, member A	-19.13	No
PKNOX2	PBX/knotted 1 homeobox 2	-18.79	No
CHP2	Calcineurin-like EF-hand protein 2	-17.99	Yes
CAMP	Cathelicidin antimicrobial peptide	-17.759	Yes^†
SERPINB4	Serpin peptidase inhibitor, clade B (ovalbumin), member 4	-17.099	Yes
SERPINB3	Serpin peptidase inhibitor, clade B (ovalbumin), member 3	-16.55	Yes^†
CAPS	Calcyphosine	-14.89	Yes^†
SCGB1D2	Secretoglobin, family 1D, member 2	-14.83	Yes^†
HS6ST3	Heparan sulfate 6-O-sulfotransferase 3	-13.98	Yes^†
PLA2G4D	Phospholipase A2, group IVD (cytosolic)	-13.79	Yes
KRT32	Keratin 32	-13.72	No
SPINK6	Serine peptidase inhibitor, Kazal type 6	-12.23	Yes
HMP19	HMP19 protein	-12.20	Yes
KRTDAP	Keratinocyte differentiation-associated protein	-11.97	Yes
HIST2H3A	Histone cluster 2, H3a	-11.97	Yes
C9orf169	Cysteine-rich tail protein 1	-11.867	No
SIM2	Single-minded family bHLH transcription factor 2	-11.857	Yes^†
DAPL1	Death-associated protein-like 1	-11.237	Yes
FABP6	Fatty acid binding protein 6, ileal	-11.17	Yes
SNORA74A	Small nucleolar RNA, H/ACA box 74A	-11.16	Yes
SNORA53	Small nucleolar RNA, H/ACA box 53	-11.12	No
CEACAM3	Carcinoembryonic antigen-related cell adhesion molecule 3	-11.06	Yes
DEFB1	Defensin, β1	-11.03	Yes
SNORA28	Small nucleolar RNA, H/ACA box 28	-10.45	No
S100A14	S100 calcium-binding protein A14	-10.39	Yes^‡
SLURP1	Secreted LY6/PLAUR domain containing 1	-10.31	Yes^‡
RGS20	Regulator of G-protein signaling 20	-10.23	Yes^‡
CEACAM5	Carcinoembryonic antigen-related cell adhesion molecule 5	-10.21	Yes^‡
HHIPL2	HHIP-like 2	-10.19	Yes
TMEM45A	Transmembrane protein 45A	-10.14	Yes^†
RN7SK	RNA, 7SK small nuclear	-10.11	No
DEFB4A	Defensin, β4A	-9.81	Yes^†
SNORD3B-1	Small nucleolar RNA, C/D box 3B-1	-9.76	No
CDSN	Corneodesmosin	-9.56	No
RAET1E	Retinoic acid early transcript 1E	-9.54	Yes^†
PVALB	Parvalbumin	-9.49	Yes
DMKN	Dermokine	-9.44	Yes
ACOX2	Acyl-CoA oxidase 2, branched chain	-9.35	Yes^†
PCDHB5	Protocadherin β 5	-9.305	Yes

^†Associated with breast cancer.

^‡Associated with triple-negative breast cancer.

Figure 3.  Protein expression of SOHLH2, DNAJC12, and LIM1 studied by immnohistochemistry on tissue microarrays containing TNBC samples.

SOHLH2 protein showed nuclear and cytoplasmic staining: (A) tumor cells with cytoplasmic weak staining. (B) Nuclear and cytoplasmic positive staining of tumor cells with a discreet stromal staining. DNAJC12 protein showed cytoplasmic staining. (C) Negative staining. (D) Cytoplasmic positive staining. LIM1 protein showed cytoplasmic staining. (E) Negative staining. (F) Positive staining.

Magnification: 20x.

Figure 4.  Downregulation of SPARC is associated with poor prognosis of TNBC.

Kaplan-Meier curves for disease-free (A) and overall (B) survival of triple-negative breast cancer patients, stratified according to SPARC protein expression. Patients were categorized as positive (moderate or intense) or negative (negative or weak) according to SPARC protein immunostaining. LogRank test was performed for curves comparison.

Figure 5.  Downregulation of SOHLH2 is associated with a worse clinical evolution of triple-negative breast cancer.

Kaplan-Meier curves for disease-free (A and C) and overall (B and D) survival of triple-negative breast cancer patients stratified according to SOHLH2 protein expression. Patients were categorized as positive (moderate orintense) or negative (negative or weak) according to SOHLH2 protein immunostaining. In (C) and (D), patients were classified in four categories according to the protein immunostaining of SPARC and SOHLH2 status. LogRank test was performed for curves comparison.

Figure 6.  Upregulation of DNAJC12 is associated with poor prognosis of triple-negative breast cancer.

Kaplan-Meier curves for disease-free (A and C) and overall (B and D) survival of triple-negative breast cancer patients stratified according to DNAJC12 protein expression. Patients were categorized as positive (>10%) ornegative (<10%) according to the percentage of tumor cells positive to DNAJC12. In (C) and (D), patients were classified in four categories according to the protein immunostaining of SPARC and DNAJC12 status. LogRank test was performed for curves comparison.

Figure 7.  LIM1 overexpression tends to be associated with more aggressive biological behavior in triple-negative breast cancer.

Kaplan-Meier curves for disease-free (A and C) and overall (B and D) survival of triple-negative breast cancer patients stratified according to LIM1 protein expression. Patients were categorized as positive (>10%) or negative (<10%) according to the percentage of tumor cells positive to LIM1. In (C) and (D), patients were classified in four categories according to the protein immunostaining of SPARC and LIM1 status. LogRank test was performed for curves comparison.