Intrinsic molecular subtypes of breast cancers categorized as HER2-positive using an alternative chromosome 17 probe assay

Abstract The 2013 update of the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) human epidermal growth factor receptor 2 (HER2) testing guidelines recommend using an alternative chromosome 17 probe assay to resolve HER2 results determined to be equivocal by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH). However, it is unclear if cases considered HER2-positive (HER2+) by the alternative probe method are similar to those classified as HER2+ by traditional IHC and FISH criteria and benefit the same from HER2-targeted therapies. We studied the clinical and pathologic features of all 31 breast cancers classified as HER2+ by the alternative probe method at our institution since 2013 and determined their PAM50 intrinsic molecular subtypes. For comparison, we analyzed 19 consecutive cases that were classified as HER2+ by traditional FISH criteria during the same time period. Thirty (97%) cancers in the alternative probe cohort were estrogen receptor (ER)-positive (ER+), while only 9/19 (47%) of traditional HER2 controls were ER+ (p = 0.0002). Sufficient tissue for intrinsic subtype analysis was available for 20/31 cancers in the alternative probe cohort and 9/19 in the traditional HER2+ group. None (0%) of the 20 alternative probe-positive cases were of the HER2-enriched intrinsic subtype, while 8/9 (89%) of those HER2+ by traditional FISH criteria were HER2-enriched (p = 0.0001). These findings suggest that breast cancers classified as HER2+ only by the alternative probe method are biologically distinct from those classified as HER2+ by traditional criteria, and raises questions as to whether or not they derive the same benefit from HER2-targeted therapies

BRG1 and BRM SWI/SNF ATPases redundantly maintain cardiomyocyte homeostasis by regulating cardiomyocyte mitophagy and mitochondrial dynamics in vivo

There has been an increasing recognition that mitochondrial perturbations play a central role in human heart failure. Discovery of mitochondrial networks, whose function is to maintain the regulation of mitochondrial biogenesis, autophagy (‘mitophagy’) and mitochondrial fusion/fission, are new potential therapeutic targets. Yet our understanding of how the molecular underpinning of these processes is just emerging. We recently identified a role of the SWI/SNF ATP-dependent chromatin remodeling complexes in the metabolic homeostasis of the adult cardiomyocyte using cardiomyocyte-specific and inducible deletion of the SWI/SNF ATPases BRG1 and BRM in adult mice (Brg1/Brm double mutant mice). To build upon these observations in early alterated metabolism, the present study looks at the subsequent alterations in mitochondrial quality control mechanisms in the impaired adult cardiomyocyte. We identified that Brg1/Brm double-mutant mice exhibited an increased mitochondrial biogenesis, increases in ‘mitophagy’, and alterations in mitochondrial fission and fusion that led to small, fragmented mitochondria. Mechanistically, increases in the autophagy and mitophagy-regulated proteins Beclin1 and Bnip3 were identified, paralleling changes seen in human heart failure. Cardiac mitochondrial dynamics were perturbed including decreased mitochondria size, reduced number, and altered expression of genes regulating fusion (Mfn1, Opa1) and fission (Drp1). We also identified cardiac protein amyloid accumulation (aggregated fibrils) during disease progression along with an increase in pre-amyloid oligomers and an upregulated unfolded protein response including increased GRP78, CHOP, and IRE-1 signaling. Together, these findings described a role for BRG1 and BRM in mitochondrial quality control, by regulating mitochondrial number, mitophagy, and mitochondrial dynamics not previously recognized in the adult cardiomyocyte. As epigenetic mechanisms are critical to the pathogenesis of heart failure, these novel pathways identified indicate that SWI/SNF chromatin remodeling functions are closely linked to mitochondrial quality control mechanisms

SWI/SNF complexes are required for full activation of the DNA-damage response

SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function

BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer

Intrinsic molecular subtypes of breast cancers categorized as HER2-positive using an alternative chromosome 17 probe assay

Abstract The 2013 update of the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) human epidermal growth factor receptor 2 (HER2) testing guidelines recommend using an alternative chromosome 17 probe assay to resolve HER2 results determined to be equivocal by immunohistochemistry (IHC) or fluorescence in-situ hybridization (FISH). However, it is unclear if cases considered HER2-positive (HER2+) by the alternative probe method are similar to those classified as HER2+ by traditional IHC and FISH criteria and benefit the same from HER2-targeted therapies. We studied the clinical and pathologic features of all 31 breast cancers classified as HER2+ by the alternative probe method at our institution since 2013 and determined their PAM50 intrinsic molecular subtypes. For comparison, we analyzed 19 consecutive cases that were classified as HER2+ by traditional FISH criteria during the same time period. Thirty (97%) cancers in the alternative probe cohort were estrogen receptor (ER)-positive (ER+), while only 9/19 (47%) of traditional HER2 controls were ER+ (p = 0.0002). Sufficient tissue for intrinsic subtype analysis was available for 20/31 cancers in the alternative probe cohort and 9/19 in the traditional HER2+ group. None (0%) of the 20 alternative probe-positive cases were of the HER2-enriched intrinsic subtype, while 8/9 (89%) of those HER2+ by traditional FISH criteria were HER2-enriched (p = 0.0001). These findings suggest that breast cancers classified as HER2+ only by the alternative probe method are biologically distinct from those classified as HER2+ by traditional criteria, and raises questions as to whether or not they derive the same benefit from HER2-targeted therapies

The BRG1 Chromatin Remodeler Protects Against Ovarian Cysts, Uterine Tumors, and Mammary Tumors in a Lineage-Specific Manner

<div><p>The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of <em>Brg1</em> null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (<em>Brg1^null/+</em>) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized <em>Wap-Cre</em> to mutate both <em>Brg1</em> floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that <em>Brg1^Wap-Cre</em> conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a <em>Brm</em>-deficient background. However, <em>Brg1^Wap-Cre</em> mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express <em>Brg1</em> and <em>Wap</em>. Thus, tumor formation in <em>Brg1</em> mutant mice appears to be confined to particular cell types that require BRG1 and also express <em>Wap</em>. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent <em>ARID1A/BAF250a</em> mutations in human ovarian and endometrial tumors.</p> </div

Expression of <i>Brg1</i>, <i>Brm</i>, and <i>Wap</i> in CD45⁻Ter119⁻CD31⁻ mammary gland subpopulations.

<p>A. Representative FACS plot of the subpopulations remaining after removing the hematopoietic and endothelial (CD45⁺Ter119⁺CD31⁺) cells from a suspension prepared from dissociated mammary tissue from a normal adult virgin mouse. The CD24⁺CD49f^low/− subset is enriched in luminal (Lum) cells; the CD24⁺CD49f⁺ subset is enriched in mature myoepithelial (Myo) cells; and the CD24⁺CD49^high subset is enriched in mammary stem cells (referred to as mammary repopulating units or MRU). B, C. RT-qPCR analysis of <i>Brg1</i> (B) and <i>Brm</i> (C) mRNA levels normalized to <i>Gapdh</i> levels in wild-type, flow-sorted mammary cell populations from adult virgin female mice. The Lum, Myo, and MRU subsets are as defined in panel A. Each histogram represents the mean ± SE from 3 independent experiments. D. RT-qPCR analysis of <i>Wap</i> mRNA levels normalized to <i>Gapdh</i> mRNA levels measured in the same 3 subsets. Lum, Myo, and MRU cells were isolated from pregnant females (E17.5 and E13.5) and virgin/nulliparous females (for representative FACS plots, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031346#pone-0031346-g001" target="_blank">Figure 1</a>). Asterisks indicate that the <i>Wap</i> signal was below the limit of detection. Each histogram represents the mean ± SE from 2 (E17.5) or 3 (E13.5, virgin/nulliparous) independent experiments.</p

Phenotype of <i>Brg1^Wap-Cre</i> mutant mice.

<p>*First pregnancy was at 2–3 months of age, and all mice were analyzed at 15–19 months of age.</p>1<p>Tg was hemizygous in each case (<i>Wap-Cre^+/0</i>).</p>2<p>Mutant genotypically but considered a control because Cre transgene expression is induced by pregnancy, which does not occur in males.</p>3<p>Did not score for ovarian or uterine phenotype.</p

Lineage-specific deletion of <i>Brg1</i> in the mammary gland.

<p>A. Whole-mount preparation of control (top) and mutant (bottom) mammary glands from multiparous females. Asterisks, lymph nodes. B. Ethidium bromide-stained gels showing <i>Brg1</i> Δfl (top) and fl (bottom) PCR products. MW, molecular-weight standard (500-, 400-, 300-, 200-, and 75-bp fragments are visible); MG, mammary gland; NP, nulliparous; MP, multiparous. C. Mammary gland section from multiparous mouse carrying the <i>Wap-Cre</i> transgene on a <i>R26R</i> background. Cre activity, visualized as blue X-Gal staining, is restricted to luminal cells (arrowhead). Basal/myoepithelial cells (arrow) are negative and appear pink because of nuclear fast red counterstain. D, E. IHC staining of BRG1 showing strong staining in nuclei throughout the mammary gland in controls (D) but absent in luminal cells of mutant mice (E). Arrows, luminal cells; asterisks, adipocyte nuclei. 400× magnification.</p