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Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach.

Beretov J, Wasinger VC, Millar EK, Schwartz P, Graham PH, Li Y - PLoS ONE (2015)

Bottom Line: Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance.Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC.Validation with a larger independent cohort of patients is required in the following study.

View Article: PubMed Central - PubMed

Affiliation: Cancer Care Centre, St George Hospital, Kogarah, Australia.

ABSTRACT

Introduction: Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method: We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results: Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions: Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.

No MeSH data available.


Related in: MedlinePlus

Validation of identified potential urine proteins ECM1, MAST4 and filaggrin from BC patients in human BC cell lines and protein MAST 4 in primary BC tissues.A. High level of MAST4 was found in the primary BC cell line (BT474) and medium levels of MAST4 were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3). High levels of ECM1 and filaggrin were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3) while low level of ECM1was seen in the primary BC cell line (BT474) and no filaggrin expression was detected in the primary BC cell line (BT474). GAPDH was used as a loading control. B. Illustration of the positive expression of MAST4 in BC using immunohistochemistry. Moderate cytoplasmic expression of MAST4 (++) were seen in the primary IBC (B1) and DCIS (B4) tumours (n = 5 for each stage of BC); there was no expression of MAST4 in the negative controls for either IBC (B2) or DCIS (B5). No staining was seen for MAST4 in normal breast tissues (B3 and B6), (n = 5). Magnifications x 400 in B1, B2, B4 and B5; magnifications x 200 in B3; magnifications x 100 in B6. Brown indicates positive staining and blue indicates nuclei. All results were from 3 independent experiments (n = 3).
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pone.0141876.g005: Validation of identified potential urine proteins ECM1, MAST4 and filaggrin from BC patients in human BC cell lines and protein MAST 4 in primary BC tissues.A. High level of MAST4 was found in the primary BC cell line (BT474) and medium levels of MAST4 were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3). High levels of ECM1 and filaggrin were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3) while low level of ECM1was seen in the primary BC cell line (BT474) and no filaggrin expression was detected in the primary BC cell line (BT474). GAPDH was used as a loading control. B. Illustration of the positive expression of MAST4 in BC using immunohistochemistry. Moderate cytoplasmic expression of MAST4 (++) were seen in the primary IBC (B1) and DCIS (B4) tumours (n = 5 for each stage of BC); there was no expression of MAST4 in the negative controls for either IBC (B2) or DCIS (B5). No staining was seen for MAST4 in normal breast tissues (B3 and B6), (n = 5). Magnifications x 400 in B1, B2, B4 and B5; magnifications x 200 in B3; magnifications x 100 in B6. Brown indicates positive staining and blue indicates nuclei. All results were from 3 independent experiments (n = 3).

Mentions: To find an association of identified potential urine protein with human BC, one existing marker-ECM1 and another two selected novel protein markers MAST4, and filaggrin were evaluated in human primary BC cell line (BT474) and metastatic BC cell lines (MDA-MB-231, MCF-7 and SK-BR-3) by Western blotting. As shown in Fig 5A, ECM1 and MAST4 were positive in all 4 BC cell lines and filaggrin was positive in the 3 metastatic BC cell lines, suggesting the identified potential urine markers from BC patients, are closely associated with human BC.


Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach.

Beretov J, Wasinger VC, Millar EK, Schwartz P, Graham PH, Li Y - PLoS ONE (2015)

Validation of identified potential urine proteins ECM1, MAST4 and filaggrin from BC patients in human BC cell lines and protein MAST 4 in primary BC tissues.A. High level of MAST4 was found in the primary BC cell line (BT474) and medium levels of MAST4 were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3). High levels of ECM1 and filaggrin were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3) while low level of ECM1was seen in the primary BC cell line (BT474) and no filaggrin expression was detected in the primary BC cell line (BT474). GAPDH was used as a loading control. B. Illustration of the positive expression of MAST4 in BC using immunohistochemistry. Moderate cytoplasmic expression of MAST4 (++) were seen in the primary IBC (B1) and DCIS (B4) tumours (n = 5 for each stage of BC); there was no expression of MAST4 in the negative controls for either IBC (B2) or DCIS (B5). No staining was seen for MAST4 in normal breast tissues (B3 and B6), (n = 5). Magnifications x 400 in B1, B2, B4 and B5; magnifications x 200 in B3; magnifications x 100 in B6. Brown indicates positive staining and blue indicates nuclei. All results were from 3 independent experiments (n = 3).
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pone.0141876.g005: Validation of identified potential urine proteins ECM1, MAST4 and filaggrin from BC patients in human BC cell lines and protein MAST 4 in primary BC tissues.A. High level of MAST4 was found in the primary BC cell line (BT474) and medium levels of MAST4 were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3). High levels of ECM1 and filaggrin were found in the metastatic BC cell lines (MDA-MB231, MCF-7 and SKBR-3) while low level of ECM1was seen in the primary BC cell line (BT474) and no filaggrin expression was detected in the primary BC cell line (BT474). GAPDH was used as a loading control. B. Illustration of the positive expression of MAST4 in BC using immunohistochemistry. Moderate cytoplasmic expression of MAST4 (++) were seen in the primary IBC (B1) and DCIS (B4) tumours (n = 5 for each stage of BC); there was no expression of MAST4 in the negative controls for either IBC (B2) or DCIS (B5). No staining was seen for MAST4 in normal breast tissues (B3 and B6), (n = 5). Magnifications x 400 in B1, B2, B4 and B5; magnifications x 200 in B3; magnifications x 100 in B6. Brown indicates positive staining and blue indicates nuclei. All results were from 3 independent experiments (n = 3).
Mentions: To find an association of identified potential urine protein with human BC, one existing marker-ECM1 and another two selected novel protein markers MAST4, and filaggrin were evaluated in human primary BC cell line (BT474) and metastatic BC cell lines (MDA-MB-231, MCF-7 and SK-BR-3) by Western blotting. As shown in Fig 5A, ECM1 and MAST4 were positive in all 4 BC cell lines and filaggrin was positive in the 3 metastatic BC cell lines, suggesting the identified potential urine markers from BC patients, are closely associated with human BC.

Bottom Line: Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance.Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC.Validation with a larger independent cohort of patients is required in the following study.

View Article: PubMed Central - PubMed

Affiliation: Cancer Care Centre, St George Hospital, Kogarah, Australia.

ABSTRACT

Introduction: Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method: We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results: Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions: Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.

No MeSH data available.


Related in: MedlinePlus