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Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers.

Manzano RG, Martinez-Navarro EM, Forteza J, Brugarolas A - Int. J. Oncol. (2014)

Bottom Line: Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4.ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance.Further studies of the most relevant phosphatases found in this study are warranted.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Genomics Laboratory, Plataforma de Oncologia, Hospital Quiron Torrevieja, 03184 Torrevieja, Alicante, Spain.

ABSTRACT
Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER-) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2+ and ERBB2- in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2+ subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.

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(A) Unsupervised hierarchical clustering (using euclidean distance metric and complete linkage) of the 41 samples from our ER− BC patients, showing the ERBB2-enriched and the basal-like enriched clusters. (B) Plot of the first 2 principal components in our ER− BC microarrays series.
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f1-ijo-45-06-2250: (A) Unsupervised hierarchical clustering (using euclidean distance metric and complete linkage) of the 41 samples from our ER− BC patients, showing the ERBB2-enriched and the basal-like enriched clusters. (B) Plot of the first 2 principal components in our ER− BC microarrays series.

Mentions: Since the seminal study by Perou et al (16) describing the different molecular BC subtypes by using expression micro-arrays, it was noted that hierarchical clustering of ER− tumors with the intrinsic signature genes yielded at least two clusters, one of them enriched in ERBB2 overexpressing tumors and another comprising mainly basal-like tumors. Although we applied a single sample predictor to the samples of our series using the classifier PAM50 published by Parker et al (17), with the exception of the basal-like subtype, the rest of the molecular subtypes did not have sufficient number of cases to analyze them separately (data not shown). Thus, we decided to apply hierarchical clustering to our samples using all the probes that in our microarray platform matched the intrinsic signature as reported by Hu et al (18). As expected, and noted previously by Perou and et al (16), mainly two clusters could be readily identified: the first was enriched in clinical ERBB2, containing 11 out of 14 (78%) of the clinical ERBB2 tumors (Fig. 1A), constituting the ERBB2-enriched molecular subtype; the second cluster contained the basal-like enriched tumors comprising TN (24 patients) and 3 clinical ERBB2 tumors. We designated the tumors belonging to the ERBB2 enriched cluster as molecular ERBB2 to distinguish them from the clinical ERBB2 (those tumors overexpressing ERBB2 by IHC), and the cluster of the predominant TN tumors as the basal-like enriched tumors even though there is a significant overlap among the clinical and molecular subtypes. We also used all the genes in the microarray to plot the first two principal components, showing that the same two groups characterized by hierarchical clustering can also be observed with a different unsupervised analysis (Fig. 1B). Given the reproducibility of this molecular classification of ER− tumors we used it to validate further our results as we did not find other large breast cancer microarray series providing information regarding the ERBB2 status as determined by IHC. SAM analysis was also applied at a 5% FDR (q<0.05) to our series to identify differentially expressed phosphatases between the molecular ERBB2 and the basal-like enriched tumors. Forty-one probes (corresponding to 38 genes) were differentially expressed (Table III). Comparing with the phosphatases identified earlier in the comparison of clinical ERBB2 with TN tumors, 23 out of 41 (56%) probes (corresponding to 20 out of 38 genes) were common with the previous analysis (Tables II and III).


Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers.

Manzano RG, Martinez-Navarro EM, Forteza J, Brugarolas A - Int. J. Oncol. (2014)

(A) Unsupervised hierarchical clustering (using euclidean distance metric and complete linkage) of the 41 samples from our ER− BC patients, showing the ERBB2-enriched and the basal-like enriched clusters. (B) Plot of the first 2 principal components in our ER− BC microarrays series.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215587&req=5

f1-ijo-45-06-2250: (A) Unsupervised hierarchical clustering (using euclidean distance metric and complete linkage) of the 41 samples from our ER− BC patients, showing the ERBB2-enriched and the basal-like enriched clusters. (B) Plot of the first 2 principal components in our ER− BC microarrays series.
Mentions: Since the seminal study by Perou et al (16) describing the different molecular BC subtypes by using expression micro-arrays, it was noted that hierarchical clustering of ER− tumors with the intrinsic signature genes yielded at least two clusters, one of them enriched in ERBB2 overexpressing tumors and another comprising mainly basal-like tumors. Although we applied a single sample predictor to the samples of our series using the classifier PAM50 published by Parker et al (17), with the exception of the basal-like subtype, the rest of the molecular subtypes did not have sufficient number of cases to analyze them separately (data not shown). Thus, we decided to apply hierarchical clustering to our samples using all the probes that in our microarray platform matched the intrinsic signature as reported by Hu et al (18). As expected, and noted previously by Perou and et al (16), mainly two clusters could be readily identified: the first was enriched in clinical ERBB2, containing 11 out of 14 (78%) of the clinical ERBB2 tumors (Fig. 1A), constituting the ERBB2-enriched molecular subtype; the second cluster contained the basal-like enriched tumors comprising TN (24 patients) and 3 clinical ERBB2 tumors. We designated the tumors belonging to the ERBB2 enriched cluster as molecular ERBB2 to distinguish them from the clinical ERBB2 (those tumors overexpressing ERBB2 by IHC), and the cluster of the predominant TN tumors as the basal-like enriched tumors even though there is a significant overlap among the clinical and molecular subtypes. We also used all the genes in the microarray to plot the first two principal components, showing that the same two groups characterized by hierarchical clustering can also be observed with a different unsupervised analysis (Fig. 1B). Given the reproducibility of this molecular classification of ER− tumors we used it to validate further our results as we did not find other large breast cancer microarray series providing information regarding the ERBB2 status as determined by IHC. SAM analysis was also applied at a 5% FDR (q<0.05) to our series to identify differentially expressed phosphatases between the molecular ERBB2 and the basal-like enriched tumors. Forty-one probes (corresponding to 38 genes) were differentially expressed (Table III). Comparing with the phosphatases identified earlier in the comparison of clinical ERBB2 with TN tumors, 23 out of 41 (56%) probes (corresponding to 20 out of 38 genes) were common with the previous analysis (Tables II and III).

Bottom Line: Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4.ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance.Further studies of the most relevant phosphatases found in this study are warranted.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics and Genomics Laboratory, Plataforma de Oncologia, Hospital Quiron Torrevieja, 03184 Torrevieja, Alicante, Spain.

ABSTRACT
Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER-) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2+ and ERBB2- in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2+ subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.

Show MeSH
Related in: MedlinePlus