This set of genes included: GATA binding protein 3 (GATA3), Netrin-4 (NTN4), Solute carrier family 7 member 8 (SLC7A8), Melanophilin (MLPH), Ectonucleotide pyrophosphates/phosphodiesterase inhibitor expert 1 (ENPP1), Laminin beta-2 chain precursor (LAMB2), and Plasminogen activator, tissue (PLAT). Since the dysregulation of GATA3 to ER�� (+) status has been extensively studied,17,18 it was chosen as an ��experimental validation control��. Two complementary approaches were adopted to validate the data obtained using microarray analysis: RT-qPCR (mRNA expression) and immunohistochemistry (protein expression). RT-qPCR analysis of the above 7 transcripts was performed in 31 tumors (for which microarray analyses were conducted) and in an independent set of 45 invasive ductal breast carcinomas.

In agreement with our microarray analysis, we detected statistically significant over-expression of all the 7 genes in ER�� (+) breast tumors as compared to ER�� (?) breast tumors: GATA3 (P < 0.0001), NTN4 (P < 0.0001), SLC7A8 (P < 0.0001), MLPH (P < 0.0001), ENPP1 (P < 0.0001), LAMB2 (P = 0.0006), and PLAT (P = 0.003) (Fig. 3). Figure 3. RT-qPCR validation of seven over expressed genes in 76 invasive breast carcinomas. Results were expressed as mean �� 2 standard error based on Log2 transformation of normalized RT-qPCR values of the assayed genes. GAPDH gene was used as normalization ... Immunohistochemistry analysis of NTN4, SLC7A8, MLPH and ENPP1 Immunohistochemistry analysis was carried out to validate our data using tumor tissues from an independent set of patient cohort, obtained as tissue microarray slides from US biomax due to non-availability of paraffin blocks for our patient cohorts.

We studied the protein expression for NTN4, SLC7A8, MLPH, ENPP1 and their ability to discriminate ER�� (+) and ER�� (?) breast tumors. As shown in Figure 4, tumor tissues from ER�� (+) group showed strong staining for our selected proteins as compared to weak staining associated with ER�� (?) group. Furthermore, unpaired t-test demonstrated statistically significant difference in the expression levels of these proteins in ER�� (+) and ER�� (?) breast cancers: NTN4 (P < 0.0009), SLC7A8 (P < 0.007), MLPH (P < 0.0001), and ENPP1 (P < 0.0147). Figure 4. IHC staining for the expression of A) NTN4 (P < 0.0009); B) SLC7A8 (P < 0.007); C) MLPH (P < 0.0001); and D) ENPP1 (P < 0.0147) in breast invasive ductal carcinoma. The left panel shows ER�� positive tissue and right ... Estrogen regulates the mRNA expression of SLC7A8, ENPP1, LAMB2 and PLAT in T-47D breast cancer cells Further we investigated the estrogen Batimastat dependant expression of NTN4, SLC7A8, MLPH, ENPP1, LAMB2, and PLAT in ER�� (+) breast cancer.