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Targeting a cell state common to triple-negative breast cancers.

Muellner MK, Mair B, Ibrahim Y, Kerzendorfer C, Lechtermann H, Trefzer C, Klepsch F, Müller AC, Leitner E, Macho-Maschler S, Superti-Furga G, Bennett KL, Baselga J, Rix U, Kubicek S, Colinge J, Serra V, Nijman SM - Mol. Syst. Biol. (2015)

Bottom Line: We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC.Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells.This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus

STAT3 is a critical downstream SYK effectorA Quantitative phosphoproteomics of MDA-MB-468 cells treated with indicated SYK inhibitors or vehicle for 6 h. Fold change in relative phosphopeptide abundance compared to DMSO is indicated.B Western blot of MDA-MB-468 cells treated as in (A).C MDA-MB-468, HCC70, DU4475 (basal) or ZR-75-1, and ZR-75-30 (luminal) cells infected with STAT3 shRNA or non-targeting control shRNA were grown for 4 days, and cell viability was measured via Celltiter Glo. **P < 0.01, ***P < 0.01, two-sided Student's t-test. n = 3.D MDA-MB-468 cells treated in dose–response matrix with SYK inhibitor R406 and two different AURKA inhibitors (AT9283 and VX680). Relative cell viability (left) and synergy as expressed by deviation from the Bliss independence model (right) are indicated.
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fig06: STAT3 is a critical downstream SYK effectorA Quantitative phosphoproteomics of MDA-MB-468 cells treated with indicated SYK inhibitors or vehicle for 6 h. Fold change in relative phosphopeptide abundance compared to DMSO is indicated.B Western blot of MDA-MB-468 cells treated as in (A).C MDA-MB-468, HCC70, DU4475 (basal) or ZR-75-1, and ZR-75-30 (luminal) cells infected with STAT3 shRNA or non-targeting control shRNA were grown for 4 days, and cell viability was measured via Celltiter Glo. **P < 0.01, ***P < 0.01, two-sided Student's t-test. n = 3.D MDA-MB-468 cells treated in dose–response matrix with SYK inhibitor R406 and two different AURKA inhibitors (AT9283 and VX680). Relative cell viability (left) and synergy as expressed by deviation from the Bliss independence model (right) are indicated.

Mentions: To gain insight into the molecular mechanism underlying the critical requirement for SYK activity in basal-like TNBCs, we determined the changes in protein tyrosine phosphorylation by quantitative phosphoproteomics upon chemical inhibition of SYK. MDA-MB-468 cells were exposed to vehicle (DMSO), R406 or BAY61-3606 for 6 h in duplicates. Following cell lysis and protein digestion, tyrosine-phosphorylated proteins were enriched. Next, samples were labeled with isobaric tandem mass tags (TMT, 6-plex) and analyzed by liquid chromatography mass spectrometry (Supplementary Table S4). Notably, a peptide corresponding to the transcription factor STAT3 with a phosphorylated tyrosine residue at position 705 was strongly reduced in the SYK inhibitor-treated samples (Fig 6A and Supplementary Fig S16). Tyr705 phosphorylation of STAT3 is required for its dimerization, nuclear translocation and DNA binding (Bromberg et al, 1999), indicating that SYK inhibitor-treated cells have reduced activity of STAT3. Inhibition of STAT3 phosphorylation upon SYK inhibition was confirmed by Western blot using a phospho-specific STAT3-Tyr705 antibody (Fig 6B). Finally, we investigated whether STAT3 down-regulation is sufficient to inhibit basal-like breast cancer cell viability. As expected, transduction of the basal-like MDA-MB-468, DU4475 and HCC70 cell lines with a shRNA targeting STAT3 resulted in a pronounced reduction of cell viability (Fig 6C and Supplementary Fig S17). In contrast, knockdown of STAT3 in the luminal cell lines ZR-75-1 and ZR-75-30, which do not respond to PKC412 and SYK inhibitors, also did not show this effect.


Targeting a cell state common to triple-negative breast cancers.

Muellner MK, Mair B, Ibrahim Y, Kerzendorfer C, Lechtermann H, Trefzer C, Klepsch F, Müller AC, Leitner E, Macho-Maschler S, Superti-Furga G, Bennett KL, Baselga J, Rix U, Kubicek S, Colinge J, Serra V, Nijman SM - Mol. Syst. Biol. (2015)

STAT3 is a critical downstream SYK effectorA Quantitative phosphoproteomics of MDA-MB-468 cells treated with indicated SYK inhibitors or vehicle for 6 h. Fold change in relative phosphopeptide abundance compared to DMSO is indicated.B Western blot of MDA-MB-468 cells treated as in (A).C MDA-MB-468, HCC70, DU4475 (basal) or ZR-75-1, and ZR-75-30 (luminal) cells infected with STAT3 shRNA or non-targeting control shRNA were grown for 4 days, and cell viability was measured via Celltiter Glo. **P < 0.01, ***P < 0.01, two-sided Student's t-test. n = 3.D MDA-MB-468 cells treated in dose–response matrix with SYK inhibitor R406 and two different AURKA inhibitors (AT9283 and VX680). Relative cell viability (left) and synergy as expressed by deviation from the Bliss independence model (right) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig06: STAT3 is a critical downstream SYK effectorA Quantitative phosphoproteomics of MDA-MB-468 cells treated with indicated SYK inhibitors or vehicle for 6 h. Fold change in relative phosphopeptide abundance compared to DMSO is indicated.B Western blot of MDA-MB-468 cells treated as in (A).C MDA-MB-468, HCC70, DU4475 (basal) or ZR-75-1, and ZR-75-30 (luminal) cells infected with STAT3 shRNA or non-targeting control shRNA were grown for 4 days, and cell viability was measured via Celltiter Glo. **P < 0.01, ***P < 0.01, two-sided Student's t-test. n = 3.D MDA-MB-468 cells treated in dose–response matrix with SYK inhibitor R406 and two different AURKA inhibitors (AT9283 and VX680). Relative cell viability (left) and synergy as expressed by deviation from the Bliss independence model (right) are indicated.
Mentions: To gain insight into the molecular mechanism underlying the critical requirement for SYK activity in basal-like TNBCs, we determined the changes in protein tyrosine phosphorylation by quantitative phosphoproteomics upon chemical inhibition of SYK. MDA-MB-468 cells were exposed to vehicle (DMSO), R406 or BAY61-3606 for 6 h in duplicates. Following cell lysis and protein digestion, tyrosine-phosphorylated proteins were enriched. Next, samples were labeled with isobaric tandem mass tags (TMT, 6-plex) and analyzed by liquid chromatography mass spectrometry (Supplementary Table S4). Notably, a peptide corresponding to the transcription factor STAT3 with a phosphorylated tyrosine residue at position 705 was strongly reduced in the SYK inhibitor-treated samples (Fig 6A and Supplementary Fig S16). Tyr705 phosphorylation of STAT3 is required for its dimerization, nuclear translocation and DNA binding (Bromberg et al, 1999), indicating that SYK inhibitor-treated cells have reduced activity of STAT3. Inhibition of STAT3 phosphorylation upon SYK inhibition was confirmed by Western blot using a phospho-specific STAT3-Tyr705 antibody (Fig 6B). Finally, we investigated whether STAT3 down-regulation is sufficient to inhibit basal-like breast cancer cell viability. As expected, transduction of the basal-like MDA-MB-468, DU4475 and HCC70 cell lines with a shRNA targeting STAT3 resulted in a pronounced reduction of cell viability (Fig 6C and Supplementary Fig S17). In contrast, knockdown of STAT3 in the luminal cell lines ZR-75-1 and ZR-75-30, which do not respond to PKC412 and SYK inhibitors, also did not show this effect.

Bottom Line: We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC.Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells.This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus