Martin Shea

Requirement for Wnt3 in vertebrate axis formation

Several studies have implicated Wnt signalling in primary axis formation during vertebrate embryogenesis, yet no Wnt protein has been shown to be essential for this process. In the mouse, primitive streak formation is the first overt morphological sign of the anterior-posterior axis. Here we show that Wnt3 is expressed before gastrulation in the proximal epiblast of the egg cylinder, then is restricted to the posterior proximal epiblast and its associated visceral endoderm and subsequently to the primitive streak and mesoderm. Wnt3–/– mice develop a normal egg cylinder but do not form a primitive streak, mesoderm or node. The epiblast continues to proliferate in an undifferentiated state that lacks anterior-posterior neural patterning, but anterior visceral endoderm markers are expressed and correctly positioned. Our results suggest that regional patterning of the visceral endoderm is independent of primitive streak formation, but the subsequent establishment of anterior-posterior neural pattern in the ectoderm is dependent on derivatives of the primitive streak. These studies provide genetic proof for the requirement of Wnt3 in primary axis formation in the mouse.

Au Nanomatryoshkas as Efficient Near-Infrared Photothermal Transducers for Cancer Treatment: Benchmarking against Nanoshells

Au nanoparticles with plasmon resonances in the near-infrared (NIR) region of the spectrum efficiently convert light into heat, a property useful for the photothermal ablation of cancerous tumors subsequent to nanoparticle uptake at the tumor site. A critical aspect of this process is nanoparticle size, which influences both tumor uptake and photothermal efficiency. Here, we report a direct comparative study of ∼90 nm diameter Au nanomatryoshkas (Au/SiO2/Au) and ∼150 nm diameter Au nanoshells for photothermal therapeutic efficacy in highly aggressive triple negative breast cancer (TNBC) tumors in mice. Au nanomatryoshkas are strong light absorbers with 77% absorption efficiency, while the nanoshells are weaker absorbers with only 15% absorption efficiency. After an intravenous injection of Au nanomatryoshkas followed by a single NIR laser dose of 2 W/cm2 for 5 min, 83% of the TNBC tumor-bearing mice appeared healthy and tumor free >60 days later, while only 33% of mice treated with nanoshells survived the same period. The smaller size and larger absorption cross section of Au nanomatryoshkas combine to make this nanoparticle more effective than Au nanoshells for photothermal cancer therapy.

An epigenomic approach to therapy for tamoxifen-resistant breast cancer

Tamoxifen has been a frontline treatment for estrogen receptor alpha (ERα)-positive breast tumors in premenopausal women. However, resistance to tamoxifen occurs in many patients. ER still plays a critical role in the growth of breast cancer cells with acquired tamoxifen resistance, suggesting that ERα remains a valid target for treatment of tamoxifen-resistant (Tam-R) breast cancer. In an effort to identify novel regulators of ERα signaling, through a small-scale siRNA screen against histone methyl modifiers, we found WHSC1, a histone H3K36 methyltransferase, as a positive regulator of ERα signaling in breast cancer cells. We demonstrated that WHSC1 is recruited to the ERα gene by the BET protein BRD3/4, and facilitates ERα gene expression. The small-molecule BET protein inhibitor JQ1 potently suppressed the classic ERα signaling pathway and the growth of Tam-R breast cancer cells in culture. Using a Tam-R breast cancer xenograft mouse model, we demonstrated in vivo anti-breast cancer activity by JQ1 and a strong long-lasting effect of combination therapy with JQ1 and the ER degrader fulvestrant. Taken together, we provide evidence that the epigenomic proteins BRD3/4 and WHSC1 are essential regulators of estrogen receptor signaling and are novel therapeutic targets for treatment of Tam-R breast cancer.

Epigenetic reprogramming of HOXC10 in endocrine-resistant breast cancer.

Genome-wide screen identifies methylation of the estrogen-repressed HOXC10 gene as a determinant of resistance to aromatase inhibitors in breast cancer. Playing Tug-of-War with HOXC10 Aromatase inhibitors are drugs that prevent androgens from being converted into estrogen, and they are frequently used to treat breast cancers that express the estrogen receptor. Unfortunately, some patients’ tumors never respond to these drugs, and others gradually become resistant over time. Although the development of resistance to aromatase inhibitors has been investigated in some previous studies and some potential mechanisms have been proposed, much about this process remains unknown. Pathiraja and colleagues began by performing a genome-wide methylation screen in breast cancer cells, which identified the developmental gene HOXC10 as a target of epigenetic silencing in the context of long-term estrogen withdrawal. When HOXC10 is active, it interferes with proliferation and can stimulate apoptosis, but estrogen suppresses its activity, thereby promoting tumor growth. By decreasing estrogen production, aromatase inhibitors up-regulate HOXC10, accounting for some of their antitumor activity. However, long-term estrogen deprivation eventually has the opposite effect, leading to methylation of HOXC10 and its long-term suppression even in the absence of estrogen. These findings suggest that a rational approach for overcoming aromatase resistance in breast cancer may involve the addition of demethylating drugs to overcome the methylation of HOXC10 and take advantage of its antitumor effects, although this remains to be demonstrated directly. Resistance to aromatase inhibitors (AIs) is a major clinical problem in the treatment of estrogen receptor (ER)–positive breast cancer. In two breast cancer cell line models of AI resistance, we identified widespread DNA hyper- and hypomethylation, with enrichment for promoter hypermethylation of developmental genes. For the homeobox gene HOXC10, methylation occurred in a CpG shore, which overlapped with a functional ER binding site, causing repression of HOXC10 expression. Although short-term blockade of ER signaling caused relief of HOXC10 repression in both cell lines and breast tumors, it also resulted in concurrent recruitment of EZH2 and increased H3K27me3, ultimately transitioning to increased DNA methylation and silencing of HOXC10. Reduced HOXC10 in vitro and in xenografts resulted in decreased apoptosis and caused antiestrogen resistance. Supporting this, we used paired primary and metastatic breast cancer specimens to show that HOXC10 was reduced in tumors that recurred during AI treatment. We propose a model in which estrogen represses apoptotic and growth-inhibitory genes such as HOXC10, contributing to tumor survival, whereas AIs induce these genes to cause apoptosis and therapeutic benefit, but long-term AI treatment results in permanent repression of these genes via methylation and confers resistance. Therapies aimed at inhibiting AI-induced histone and DNA methylation may be beneficial in blocking or delaying AI resistance.

Sub-100 nm gold nanomatryoshkas improve photo-thermal therapy efficacy in large and highly aggressive triple negative breast tumors

There is an unmet need for efficient near-infrared photothermal transducers for the treatment of highly aggressive cancers and large tumors where the penetration of light can be substantially reduced, and the intra-tumoral nanoparticle transport is restricted due to the presence of hypoxic or necrotic regions. We report the performance advantages obtained by sub 100nm gold nanomatryushkas, comprising concentric gold-silica-gold layers compared to conventional ~150nm silica core gold nanoshells for photothermal therapy of triple negative breast cancer. We demonstrate that a 33% reduction in silica-core-gold-shell nanoparticle size, while retaining near-infrared plasmon resonance, and keeping the nanoparticle surface charge constant, results in a four to five fold tumor accumulation of nanoparticles following equal dose of injected gold for both sizes. The survival time of mice bearing large (>1000mm(3)) and highly aggressive triple negative breast tumors is doubled for the nanomatryushka treatment group under identical photo-thermal therapy conditions. The higher absorption cross-section of a nanomatryoshka results in a higher efficiency of photonic to thermal energy conversion and coupled with 4-5× accumulation within large tumors results in superior therapy efficacy.

The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family, defined by the adjacent, region-specific homeotic gene spalt

Abstract We report the full coding sequence of a new Drosophila gene, spalt-related, which is homologous and adjacent to the region-specific homeotic gene, spalt. Both genes have three widely spaced sets of C2H2 zinc finger motifs, but spalt-related encodes a fourth pair of C-terminal fingers resembling the Xenopus homologue, Xsal-1. The degrees of sequence divergence among all three members of this family are comparable, suggesting that the Drosophila genes originated from an ancient gene duplication. The spalt-related gene is expressed with quantitative variations from mid-embryogenesis (8–12 h) to the adult stage, but not in ovaries or early embryos. Expression is localized to limited parts of the body, including specific cell populations in the nervous system. In the wing disc, spalt and spalt-related are expressed in indistinguishable domains; in the nervous system and some other organs the expression patterns extensively overlap but are not identical, indicating that the genes have partially diverged in terms of developmental regulation. A characteristic central set of zinc fingers specifically binds to an A/T-rich consensus sequence, defining some DNA binding properties of this ancient family of nuclear factors.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase

IntroductionActivation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.MethodsAltered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.ResultsModerate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.ConclusionsModerate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer

Significance One of the mechanisms of endocrine resistance in estrogen receptor α (ER)-positive (+) breast cancer is the cross-talk between the ER and growth factor receptor pathways leading to altered ER activity and a reprogrammed ER-dependent transcriptome. However, key mediators of this ER-dependent transcriptional reprogramming remain elusive. Here we demonstrate that forkhead box protein A1 (FOXA1) up-regulation via gene amplification or overexpression contributes to endocrine resistance and increased invasiveness phenotypes by altering the ER-dependent transcriptome. We further show that IL-8, one of the top altered FOXA1/ER effectors, plays a key role in mediating these phenotypes and is a potential target to treat ER+/FOXA1-high breast cancer. Our findings provoke a new interplay of FOXA1 in the ER transcriptional program in endocrine-resistant breast cancer. Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)–chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Abstract PD01-01: Overcoming endocrine therapy resistance related to PTEN loss by strategic combinations with mTOR, AKT, or MEK inhibitors

Background: Hyperactive PI3K signaling is associated with a more aggressive subtype of estrogen receptor (ER) positive breast cancer (BC) and with endocrine resistance. Loss or downregulation of PI3K9s inhibitor PTEN is more common in basal and luminal B vs. luminal A BC. However, the role of PTEN in modulating response to various endocrine therapies is unclear. Here we investigated the effects of PTEN knockdown (KD) on endocrine sensitivity and the potential of multiple kinase inhibitors to restore and improve responses. Methods: Nude mice bearing ER+ BC xenograft tumors of MCF7 cells stably expressing a doxycycline (Dox)-inducible PTEN-shRNA were randomized to four endocrine treatment groups [continued estrogen (E2) supplementation, or E2-deprivation (ED) alone or in combination with tamoxifen (Tam) or fulvestrant (Ful)]; all -/+ Dox. The effects of single or combined kinase inhibitors on these endocrine treatments -/+ Dox were studied in vitro using inhibitors (i) to mTOR (AZD2014, 0.2 μM), AKT (AZD5363, 1 μM), or MEK (Selumetinib/ARRY-142886, 1 μM). Cell growth, apoptosis, and ER and progesterone receptor (PR) signaling were analyzed using cell cytometry, qRT/PCR, and Western blotting. Synergism tests were used to examine the growth effects of the most promising combinatorial therapy with multiple kinase inhibitors in different endocrine settings. Results: In wild-type (WT) PTEN xenograft tumors, endocrine therapies were very effective, inducing frequent tumor regression. In PTEN KD tumors endocrine therapies were less effective — PTEN KD delayed tumor regression in all endocrine regimens and accelerated tumor progression in the Tam treated group. Furthermore, at day 250, only 1/8 and 0/7 tumors had developed resistance in the ED and the Ful (−Dox) groups, respectively, while with PTEN KD (+Dox), 7/15 and 5/15 tumors developed resistance to ED and to Ful. In vitro PTEN KD also induced resistance to all endocrine therapies. mRNA and/or protein levels of ER and PR were suppressed by PTEN KD and restored by mTORi and AKTi. In cells with WT PTEN, mTORi was highly effective with or without endocrine therapy. However, AKTi and MEKi were more effective in combination with endocrine therapy. All three inhibitors were less effective upon PTEN KD. The mTORi plus AKTi combination resulted in a potent synergistic inhibition in PTEN KD cells in the presence of E2 or with ED. In contrast, in the presence of Tam, AKTi plus MEKi, independent of PTEN status, was the most effective combination at the doses chosen. Finally, these inhibitors and combinations were more effective in the presence of Ful than ED or Tam in WT PTEN cells. AKTi combined with Ful was still highly effective even in PTEN KD cells, but mTORi and MEKi were less effective. Conclusions: Our results suggest that PTEN loss renders endocrine therapy less effective in in vitro and in vivo experimental models. Single AKT/MEK kinase inhibitors are more potent in the presence of endocrine therapy. In PTEN KD cells, the activity of all three kinase inhibitors is largely diminished, except for AKTi in the presence of fulvestrant. Kinase inhibitor combinations are generally more effective, but the optimal combinations vary by PTEN status and type of endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-01.