Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.

There was an error published in Development 135, 1605-1613.The gene Brn3a was incorrectly written as Brn3b throughout, including Fig. 8G. On p. 1611, line 12, Brn3b (Pou4f2) should read Brn3a (Pou4f1).The authors apologise to readers for this mistake.

Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15–20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues.SignificanceThe matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment.