CDK7-dependent transcriptional addiction in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.

The p110α and p110ß isoforms of PI3K play divergent roles in mammary gland development and tumorigenesis.

Class Ia phosphatidylinositol 3 kinase (PI3K) is required for oncogenic receptor-mediated transformation; however, the individual roles of the two commonly expressed class Ia PI3K isoforms in oncogenic receptor signaling have not been elucidated in vivo. Here, we show that genetic ablation of p110α blocks tumor formation in both polyoma middle T antigen (MT) and HER2/Neu transgenic models of breast cancer. Surprisingly, p110ß ablation results in both increased ductal branching and tumorigenesis. Biochemical analyses suggest a competition model in which the less active p110ß competes with the more active p110α for receptor binding sites, thereby modulating the level of PI3K activity associated with activated receptors. Our findings demonstrate a novel p110ß-based regulatory role in receptor-mediated PI3K activity and identify p110α as an important target for treatment of HER2-positive disease.

Incidence of Adjacent Synchronous Invasive Carcinoma and/or Ductal Carcinoma In-situ in Patients with Lobular Neoplasia on Core Biopsy: Results from a Prospective Multi-Institutional Registry (TBCRC 020).

BACKGROUND: Lobular neoplasia (LN) represents a spectrum of atypical proliferative lesions, including atypical lobular hyperplasia and lobular carcinoma-in-situ. The need for excision for LN found on core biopsy (CB) is controversial. We conducted a prospective multi-institutional trial (TBCRC 20) to determine the rate of upgrade to cancer after excision for pure LN on CB. METHODS: Patients with a CB diagnosis of pure LN were prospectively identified and consented to excision. Cases with discordant imaging and those with additional lesions requiring excision were excluded. Upgrade rates to cancer were quantified on the basis of local and central pathology review. Confidence intervals and sample size were based on exact binomial calculations. RESULTS: A total of 77 of 79 registered patients underwent excision (median age 51 years, range 27-82 years). Two cases (3%; 95% confidence interval 0.3-9) were upgraded to cancer (one tubular carcinoma, one ductal carcinoma-in-situ) at excision per local pathology. Central pathology review of 76 cases confirmed pure LN in the CB in all but two cases. In one case, the tubular carcinoma identified at excision was also found in the CB specimen, and in the other, LN was not identified, yielding an upgrade rate of one case (1%; 95% CI 0.01-7) by central pathology review. CONCLUSIONS: In this prospective study of 77 patients with pure LN on CB, the upgrade rate was 3% by local pathology and 1% by central pathology review, demonstrating that routine excision is not indicated for patients with pure LN on CB and concordant imaging findings.

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1.

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.

X chromosomal abnormalities in basal-like human breast cancer.

Sporadic basal-like cancers (BLC) are a distinct class of human breast cancers that are phenotypically similar to BRCA1-associated cancers. Like BRCA1-deficient tumors, most BLC lack markers of a normal inactive X chromosome (Xi). Duplication of the active X chromosome and loss of Xi characterized almost half of BLC cases tested. Others contained biparental but nonheterochromatinized X chromosomes or gains of X chromosomal DNA. These abnormalities did not lead to a global increase in X chromosome transcription but were associated with overexpression of a small subset of X chromosomal genes. Other, equally aneuploid, but non-BLC rarely displayed these X chromosome abnormalities. These results suggest that X chromosome abnormalities contribute to the pathogenesis of BLC, both inherited and sporadic.

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

DNA-dependent protein kinase (DNA-PK)-dependent cisplatin-induced loss of nucleolar facilitator of chromatin transcription (FACT) and regulation of cisplatin sensitivity by DNA-PK and FACT.

Both the Ku subunit of the DNA-dependent protein kinase (DNA-PK) and the facilitator of chromatin transcription (FACT) complex reportedly bind cisplatin-DNA adducts. For this study, we developed an immunocytochemical assay based on detergent extraction allowing unveiling nucleolar subpopulations of proteins present in both the nucleoplasm and the nucleolus. Immunofluorescence analysis in various human cancer cell lines and immunoblotting of isolated nucleoli show that DNA-PK catalytic subunit (DNA-PKcs), Ku86, the Werner syndrome protein (WRN), and the structure-specific recognition protein 1 (SSRP1) subunit of FACT colocalize in the nucleolus and exit the nucleolus after cisplatin treatment. Nucleolar localization of Ku is also lost after gamma or UV irradiation and exposure to DNA-damaging drugs, such as actinomycin D, mitomycin C, hydroxyurea, and doxorubicin. Ku86 and WRN leave the nucleolus after exposure to low (>1 microg/mL) doses of cisplatin. In contrast, the SSRP1 association with the nucleolus was disrupted only by high (50-100 microg/mL) doses of cisplatin. Both cisplatin-induced loss of nucleolar SSRP1 and DNA-PK activation are suppressed by pretreatment of the cells with wortmannin or the DNA-PK inhibitor NU7026 but not by the phosphatidylinositol 3-kinase inhibitor LY294002. In the same conditions, kinase inhibitors did not alter the exit of DNA-PKcs and WRN, suggesting that different mechanisms regulate the exit of DNA-PK/WRN and FACT from the nucleolus. Furthermore, RNA silencing of DNA-PKcs blocked the cisplatin-induced exit of nucleolar SSRP1. Finally, silencing of DNA-PKcs or SSRP1 by short hairpin RNA significantly increased the sensitivity of cancer cells to cisplatin.

Molecular imaging of the breast.

OBJECTIVE: Personalized health care centers around the concept that each tumor and its host environment is unique; optimal treatment and expected response for any given woman presenting with a newly diagnosed breast cancer differ from the care and response of other women. CONCLUSION: As more is understood about the molecular subtypes of breast cancer and as development of targeted therapies progresses, the possibility of earlier treatment response assessment and even improved detection will be realized.

Circulating tumor cells in HER-2 positive metastatic breast cancer patients treated with trastuzumab and chemotherapy.

The detection of circulating tumor cells (CTCs) in peripheral blood may have important prognostic and predictive implications in breast cancer treatment. A limitation in this field has been the lack of a validated method of accurately measuring CTCs. While sensitivity has improved using RT-PCR, specificity remains a major challenge. The goal of this paper is to present a sensitive and specific methodology of detecting CTCs in women with HER-2 positive metastatic breast cancer, and to examine its role as a marker that tracks disease response during treatment with trastuzumab-containing regimens. The study included patients with HER-2-positive metastatic breast cancer enrolled on two different clinical protocols using a trastuzumab-containing regimen. Serial CTCs were measured at planned time points and clinical correlations were made. Immunomagnetic selection of circulating epithelial cells was used to address the specificity of tumor cell detection using cytokeratin 19 (CK19). In addition, the extracellular domain of the HER-2 protein (HER-2/ECD) was measured to determine if CTCs detected by CK19 accurately reflect tumor burden. The presence of CTCs at first restaging was associated with disease progression. We observed an association between CK19 and HER-2/ECD. The association of HER-2/ECD with clinical response followed a similar pattern to that seen with CK19. Finally, the absence of HER-2/ECD at best overall response and a change of HER-2/ECD from positive at baseline to negative at best overall response was associated with favorable treatment response. Our study supports the prognostic and predictive role of the detection of CTCs in treatment of HER-2-positive metastatic breast cancer patients. The association between CK19 and markers of disease burden is in line with the concept that CTCs may be a reliable measure of tumor cells in the peripheral blood of patients with metastatic breast cancer. The association of CTCs at first restaging with treatment failure indicates that CTCs may have a role as surrogate markers to monitor treatment response.

Metastatic potential of 21T human breast cancer cells depends on Akt/protein kinase B activation.

Most cancer lethality is caused by metastasis. To gain insight into the molecular basis of tumor progression to metastasis, we used the 21T series of human mammary epithelial cells obtained by successive biopsies from one breast cancer patient. The c-erbB2 gene is amplified and overexpressed in each of three 21T tumor lines. The erbB receptor tyrosine kinase-activated phosphatidylinositol 3-kinase/Akt signaling cascade is crucial for the development and maintenance of epithelial cells, and dysregulation of this pathway is frequently associated with cellular transformation and cancer. For Akt to be fully activated, Ser(473) on its COOH terminus needs to be phosphorylated. We detected more Ser(473) Akt phosphorylation in MT cells, derived from a pleural effusion, compared with cells from the primary tumor. This phosphorylation has recently been shown to be catalyzed by mammalian target of rapamycin (mTOR)/rictor kinase. By using genetic and pharmacologic activators and inhibitors, we showed that Ser(473) Akt phosphorylation is more sensitive to mTOR/rictor inhibition in metastatic tumor cells than normal mammary epithelial and primary tumor cells. The mTOR/rictor kinase activity was indispensable for both Ser(473) Akt phosphorylation and migration of metastatic MT2 cells. In addition, a large decrease of protein phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) was found, which could be responsible for the overexpression of Ser(473) Akt in MT cells. Our data indicate that these breast cancer cells acquire new vulnerabilities, rictor and PHLPP, which might provide an Achilles' heel for therapeutic intervention of breast cancer metastasis.

Risk prediction models with incomplete data with application to prediction of estrogen receptor-positive breast cancer: prospective data from the Nurses' Health Study.

INTRODUCTION: A number of breast cancer risk prediction models have been developed to provide insight into a woman's individual breast cancer risk. Although circulating levels of estradiol in postmenopausal women predict subsequent breast cancer risk, whether the addition of estradiol levels adds significantly to a model's predictive power has not previously been evaluated. METHODS: Using linear regression, the authors developed an imputed estradiol score using measured estradiol levels (the outcome) and both case status and risk factor data (for example, body mass index) from a nested case-control study conducted within a large prospective cohort study and used multiple imputation methods to develop an overall risk model including both risk factor data from the main cohort and estradiol levels from the nested case-control study. RESULTS: The authors evaluated the addition of imputed estradiol level to the previously published Rosner and Colditz log-incidence model for breast cancer risk prediction within the larger Nurses' Health Study cohort. The follow-up was from 1980 to 2000; during this time, 1,559 invasive estrogen receptor-positive breast cancer cases were confirmed. The addition of imputed estradiol levels significantly improved risk prediction; the age-specific concordance statistic increased from 0.635 +/- 0.007 to 0.645 +/- 0.007 (P < 0.001) after the addition of imputed estradiol. CONCLUSION: Circulating estradiol levels in postmenopausal women appear to add to other lifestyle factors in predicting a woman's individual risk of breast cancer.

Predictors of resistance to preoperative trastuzumab and vinorelbine for HER2-positive early breast cancer.

PURPOSE: To assess pathologic complete response (pCR), clinical response, feasibility, safety, and potential predictors of response to preoperative trastuzumab plus vinorelbine in patients with operable, human epidermal growth factor receptor 2 (HER2)-positive breast cancer. EXPERIMENTAL DESIGN: Forty-eight patients received preoperative trastuzumab and vinorelbine weekly for 12 weeks. Single and multigene biomarker studies were done in an attempt to identify predictors of response. RESULTS: Eight of 40 (20%) patients achieved pCR (95% confidence interval, 9-36%). Of 9 additional patients recruited for protocol-defined toxicity analysis, 8 were evaluable; 42 of 48 (88%) patients had clinical response (16 patients, clinical complete response; 26 patients, clinical partial response). T(1) tumors more frequently exhibited clinical complete response (P = 0.05) and showed a trend to exhibit pCR (P = 0.07). Five (13%) patients experienced grade 1 cardiac dysfunction during preoperative treatment. Neither HER2 nor estrogen receptor status changed significantly after exposure to trastuzumab and vinorelbine. RNA profiling identified three top-level clusters by unsupervised analysis. Tumors with extremes of response [pCR (n = 3) versus nonresponse (n = 3)] fell into separate groups by hierarchical clustering. No predictive genes were identified in pCR tumors. Nonresponding tumors were more likely to be T(4) stage (P = 0.02) and express basal markers (P < 0.00001), growth factors, and growth factor receptors. Insulin-like growth factor-I receptor membrane expression was associated with a lower response rate (50% versus 97%; P = 0.001). CONCLUSIONS: Preoperative trastuzumab plus vinorelbine is active and well tolerated in patients with HER2-positive, operable, stage II/III breast cancer. HER2-overexpressing tumors with a basal-like phenotype, or with expression of insulin-like growth factor-I receptor and other proteins involved in growth factor pathways, are more likely to be resistant to this regimen.

Nuclear factor-kappaB activation: a molecular therapeutic target for estrogen receptor-negative and epidermal growth factor receptor family receptor-positive human breast cancer.

Nuclear factor-kappaB (NF-kappaB), a transcription factor with pleotropic effects, is a downstream mediator of growth signaling in estrogen receptor (ER)-negative and erbB family particularly erbB2 (HER-2/neu) receptor-positive cancer. We previously reported activation of NF-kappaB in ER-negative breast cancer cells and breast tumor specimens, but the consequence of inhibiting NF-kappaB activation in this subclass of breast cancer has not been shown. In this study, we investigated the role of NF-kappaB activation by studying the tumorigenic potential of cells expressing genetically manipulated, inducible, dominant-negative inhibitory kappaB kinase (IKK) beta in xenograft tumor model. Conditional inhibition of NF-kappaB activation by the inducible expression of dominant-negative IKKbeta simultaneously blocked cell proliferation, reinstated apoptosis, and dramatically blocked xenograft tumor formation. Secondly, the humanized anti-erbB2 antibody trastuzumab (Herceptin) and the specific IKK inhibitor NF-kappaB essential modifier-binding domain peptide both blocked NF-kappaB activation and cell proliferation and reinstated apoptosis in two ER-negative and erbB2-positive human breast cancer cell lines that are used as representative model systems. Combinations of these two target-specific inhibitors synergistically blocked cell proliferation at concentrations that were singly ineffective. Inhibition of NF-kappaB activation with two other low molecular weight compounds, PS1145 and PS341, which inhibited IKK activity and proteasome-mediated phosphorylated inhibitory kappaB protein degradation, respectively, blocked erbB2-mediated cell growth and reversed antiapoptotic machinery. These results implicate NF-kappaB activation in the tumorigenesis and progression of ER-negative breast cancer. It is postulated that this transcription factor and its activation cascade offer therapeutic targets for erbB2-positive and ER-negative breast cancer.

Crossroads of estrogen receptor and NF-kappaB signaling.

Cellular homeostasis in higher organisms is maintained by balancing cell growth, differentiation, and death. Two important systems that transmit extracellular signals into the machinery of the cell nucleus are the signaling pathways that activate nuclear factor kappaB (NF-kappaB) and estrogen receptor (ER). These two transcription factors induce expression of genes that control cell fates, including proliferation and cell death (apoptosis). However, ER has anti-inflammatory effects, whereas activated NF-kappaB initiates and maintains cellular inflammatory responses. Recent investigations elucidated a nonclassical and nongenomic effect of ER: inhibition of NF-kappaB activation and the inflammatory response. In breast cancer, antiestrogen therapy might cause reactivation of NF-kappaB, potentially rerouting a proliferative signal to breast cancer cells and contributing to hormone resistance. Thus, ER ligands that selectively block NF-kappaB activation could provide specific potential therapy for hormone-resistant ER-positive breast cancers.

Rate-limiting effects of Cyclin D1 in transformation by ErbB2 predicts synergy between herceptin and flavopiridol.

Cyclin D1 is downstream of erbB2 and is required for erbB2 transformation. Here we report thatcyclin D1 functions are essential, rate limiting for erbB2 transformation, and reciprocally increase erbB2 levels. This interaction depends on three cyclin D1 activities: cyclin-dependent kinase 4-dependent kinase activity, titration of p27, and an intrinsic transcriptional activity of cyclin D1. Drugs active against erbB2 and cyclin D1 (Herceptin and flavopiridol) were synergistically cytotoxic against erbB2-positive breast cancer cell lines. Addition of flavopiridol to Herceptin synergistically lowered erbB2 levels in these cells. Our data suggest the potential use of combinations of cyclin-dependent kinase inhibitors and Herceptin in breast cancer.

Identification of genes responsible for RelA-dependent proliferation arrest in human mammary epithelial cells conditionally expressing RelA.

The molecular mechanisms responsible for opposing oncogenic and tumor-suppressor activities of NF-kB are obscure. Semi-quantitative immunohistochemistry of primary breast tumors using antibodies to RelA, the pleiotropic NF-kB factor, and Ki67 revealed a negative correlation between RelA levels and Ki67-index among ER +/HER2 - tumors [1]. Similarly, expression of AURKA, a marker for proliferation, negatively correlates with expression of NFKBIA, a surrogate for RelA expression and activity, in ER +/HER2 - tumors analyzed by The Cancer Genome Atlas [2], [3], [4]. Furthermore, conditional expression of RelA using a Tetracycline-inducible system in Human Mammary Epithelial Cells (HRA cells) caused proliferation arrest while withdrawal of Doxycycline (Dox) and suppression of RelA expression in arrested cells restored cell cycle progression [1]. To identify genes responsible for the negative relationship between RelA levels and proliferation, we performed genome-wide gene expression analysis of HRA cells under the following conditions: RelA un-induced, No Dox (ND); Dox induced for 24 h; Dox induced for 72 h; Dox induced for 24 h then Dox withdrawn for 48 h. The expression data was submitted to Gene Expression Ominibus (GEO) and the accession number is GSE65040. Analysis of the data identified cross-talk between basal RelA activity and the Interferon pathway mediated by IRF1, a target of RelA [5]. Activation of the Interferon pathway lead to down-regulation of CDK4 expression resulting in RB1 hypo-phosphorylation and suppression of cell cycle progression. The tumor-suppressor activity of NF-kB, specifically RelA, may stem from cross-talk with the Interferon pathway.

Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases.

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response was correlated with a reduction in the phosphorylation of 4EBP1, an mTORC1 effector. The two nonresponding PDXs showed hypermutated genomes with enrichment of mutations in DNA-repair genes, which suggests an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor in combination with an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.

RelA-Induced Interferon Response Negatively Regulates Proliferation.

Both oncogenic and tumor-suppressor activities are attributed to the Nuclear Factor kappa B (NF-kB) pathway. Moreover, NF-kB may positively or negatively regulate proliferation. The molecular determinants of these opposing roles of NF-kB are unclear. Using primary human mammary epithelial cells (HMEC) as a model, we show that increased RelA levels and consequent increase in basal transcriptional activity of RelA induces IRF1, a target gene. Induced IRF1 upregulates STAT1 and IRF7, and in consort, these factors induce the expression of interferon response genes. Activation of the interferon pathway down-regulates CDK4 and up-regulates p27 resulting in Rb hypo-phosphorylation and cell cycle arrest. Stimulation of HMEC with IFN-γ elicits similar phenotypic and molecular changes suggesting that basal activity of RelA and IFN-γ converge on IRF1 to regulate proliferation. The anti-proliferative RelA-IRF1-CDK4 signaling axis is retained in ER+/HER2- breast tumors analyzed by The Cancer Genome Atlas (TCGA). Using immuno-histochemical analysis of breast tumors, we confirm the negative correlation between RelA levels and proliferation rate in ER+/HER2- breast tumors. These findings attribute an anti-proliferative tumor-suppressor role to basal RelA activity. Inactivation of Rb, down-regulation of RelA or IRF1, or upregulation of CDK4 or IRF2 rescues the RelA-IRF1-CDK4 induced proliferation arrest in HMEC and are points of disruption in aggressive tumors. Activity of the RelA-IRF1-CDK4 axis may explain favorable response to CDK4/6 inhibition observed in patients with ER+ Rb competent tumors.

Pre-counseling education materials for BRCA testing: does tailoring make a difference?

Although tailored print materials (TPMs) have been assessed for a variety of behavioral targets, their effectiveness as decision aids for genetic testing had not been evaluated at the time this study began. We compared TPMs and non-tailored print material (NPMs) that included similar content about genetic testing for breast and ovarian cancer susceptibility. TPMs were prepared especially for an individual based on information from and about her. We mailed baseline surveys to 461 women referred by physicians or identified through a tumor registry. All had personal and family histories of breast and/or ovarian cancer and, on the basis of these histories, an estimated > or =10% probability of carrying a mutation in the breast/ovarian cancer genes BRCA1 or BRCA2. The 325 (70%) who responded were randomly assigned to receive TPM or NPM. Followup surveys, mailed 2 weeks following receipt of print materials, were returned by 262 women (81% of baseline responders). Participants were predominately white (94%) and well-educated (50% college graduates). The mean age was 49 years. At follow-up, TPM recipients exhibited significantly greater improvement in percent of correct responses for the 13-item true/false measure of knowledge (24% increase for TPM vs. 16% for NPM; p < 0.0001) and significantly less over-estimation of risk of being a mutation carrier (40% TPM group overestimated vs. 70% NPM; p < 0.0001). Anxiety did not differ significantly between groups. Reactions to materials differed on two items: "seemed to be prepared just for me" (76% TPM vs. 52% NPM; p < 0.001) and "told me what I wanted to know about BRCA1 and 2 testing" (98% TPM vs. 91% NPM; p < 0.05). TPMs showed an advantage in increasing knowledge and enhancing accuracy of perceived risk. Both are critical components of informed decision making.