Total Cellular ATP Production Changes With Primary Substrate in MCF7 Breast Cancer Cells.

Cancer growth is predicted to require substantial rates of substrate catabolism and ATP turnover to drive unrestricted biosynthesis and cell growth. While substrate limitation can dramatically alter cell behavior, the effects of substrate limitation on total cellular ATP production rate is poorly understood. Here, we show that MCF7 breast cancer cells, given different combinations of the common cell culture substrates glucose, glutamine, and pyruvate, display ATP production rates 1.6-fold higher than when cells are limited to each individual substrate. This increase occurred mainly through faster oxidative ATP production, with little to no increase in glycolytic ATP production. In comparison, non-transformed C2C12 myoblast cells show no change in ATP production rate when substrates are limited. In MCF7 cells, glutamine allows unexpected access to oxidative capacity that pyruvate, also a strictly oxidized substrate, does not. Pyruvate, when added with other exogenous substrates, increases substrate-driven oxidative ATP production, by increasing both ATP supply and demand. Overall, we find that MCF7 cells are highly flexible with respect to maintaining total cellular ATP production under different substrate-limited conditions, over an acute (within minutes) timeframe that is unlikely to result from more protracted (hours or more) transcription-driven changes to metabolic enzyme expression. The near-identical ATP production rates maintained by MCF7 and C2C12 cells given single substrates reveal a potential difficulty in using substrate limitation to selectively starve cancer cells of ATP. In contrast, the higher ATP production rate conferred by mixed substrates in MCF7 cells remains a potentially exploitable difference.

Chronic cadmium exposure decreases the dependency of MCF7 breast cancer cells on ERα.

Cadmium is an environmental contaminant that can activate estrogen receptor alpha (ERα) and contribute to the development and progression of breast cancer. Our lab previously demonstrated that chronic cadmium exposure alters the expression of several ERα-responsive genes and increases the malignancy of breast cancer cells. Although these studies support cadmium's function as a hormone disrupter, the role of ERα in cadmium-induced breast cancer progression remains unclear. To address this, we modulated the expression of ERα and found that while the loss of ERα significantly impaired cancer cell growth, migration, invasion and anchorage-independent growth in both MCF7 and MCF7-Cd cells, cadmium-exposed cells retained a significant advantage in cell growth, migration, and invasion, and partially circumvented the loss of ERα. ERα knockout in MCF7 and MCF7-Cd cells significantly reduced the expression of classical ERα-regulated genes, while non-classical ERα-regulated genes were less impacted by the loss of ERα in MCF7-Cd cells. This is the first study to show that chronic cadmium exposure, even at low levels, can increase the malignancy of breast cancer cells by decreasing their dependency on ERα and increasing the adaptability of the cancer cells.

Personalized Detection of Circulating Tumor DNA Antedates Breast Cancer Metastatic Recurrence.

PURPOSE: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer. EXPERIMENTAL DESIGN: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n = 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X). RESULTS: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling. CONCLUSIONS: This study demonstrates that patient-specific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention.

Novel flexible heteroarotinoid, SL-1-39, inhibits HER2-positive breast cancer cell proliferation by promoting lysosomal degradation of HER2.

SL-1-39 [1-(4-chloro-3-methylphenyl)-3-(4-nitrophenyl)thiourea] is a new flexible heteroarotinoid (Flex-Het) analog derived from the parental compound, SHetA2, previously shown to inhibit cell growth across multiple cancer types. The current study aims to determine growth inhibitory effects of SL-1-39 across the different subtypes of breast cancer cells and delineate its molecular mechanism. Our results demonstrate that while SL-1-39 blocks cell proliferation of all breast cancer subtypes tested, it has the highest efficacy against HER2+ breast cancer cells. Molecular analyses suggest that SL-1-39 prevents S phase progression of HER2+ breast cancer cells (SKBR3 and MDA-MB-453), which is consistent with reduced expression of key cell-cycle regulators at both the protein and transcriptional levels. SL-1-39 treatment also decreases the protein levels of HER2 and pHER2 as well as its downstream effectors, pMAPK and pAKT. Reduction of HER2 and pHER2 at the protein level is attributed to increased lysosomal degradation of total HER2 levels. This is the first study to show that a flexible heteroarotinoid analog modulates the HER2 signaling pathway through lysosomal degradation, and thus further warrants the development of SL-1-39 as a therapeutic option for HER2+ breast cancer.

Steroid hormone receptors as prognostic markers in breast cancer.

Despite the existence of many promising anti-cancer therapies, not all breast cancers are equally treatable, due partly to the fact that focus has been primarily on a few select breast cancer biomarkers- notably ERα, PR and HER2. In cases like triple negative breast cancer (ERα-, PR-, and HER2-), there is a complete lack of available biomarkers for prognosis and therapeutic purposes. The goal of this review is to determine if other steroid receptors, like ERß and AR, could play a prognostic and/or therapeutic role. Data from various in vitro, in vivo, and clinical breast cancer studies were examined to analyze the presence and function of ERß, PR, and AR in the presence and absence of ERα. Additionally, we focused on studies that examined how expression of the various steroid receptor isoforms affects breast cancer progression. Our findings suggest that while we have a solid understanding of how these receptors work individually, how they interact and behave in the presence and absence of other receptors requires further research. Furthermore, there is an incomplete understanding of how the various steroid receptor isoforms interact and impact receptor function and breast cancer progression, partly due to the difficulty in detecting all the various isoforms. More large-scale clinical studies must be made to analyze systematically the expression of steroid hormone receptors and their respective isoforms in breast cancer patients in order to determine how these receptors interact with each other and in turn affect cancer progression.

Novel flexible heteroarotinoid, SL-1-18, promotes ERα degradation to inhibit breast cancer cell growth.

SL-1-18 (1-(chrysen-6-yl)-3-(4-nitrophenyl)thiourea) is new flexible heteroarotinoid (Flex-Het) analog derived from the parent compound, SHetA2, and our previous study showed comparable activity to SHetA2 in terms of inhibiting ER+ breast cancer cell growth. This current study aims to determine the molecular mechanism underlying SL-1-18's effect on breast cancer cell growth. Our results indicate that SL-1-18 inhibits cell proliferation of ER+ breast cancer cells (MCF-7 and T-47D) by preventing cell cycle progression. SL-1-18 treatment correlated positively with decreased expression of key cell-cycle regulators, such as cyclin D1, as well as other ERα-target genes at both the transcript and protein levels. Interestingly, decreased expression of ERα was also observed, with a significant reduction at the protein level within 2 h of SL-1-18 treatment, while the decrease in mRNA occurred at a later time point. ERα degradation was shown to be mediated by the ubiquitination-proteasome pathway. In summary, this is the first study to show that a Flex-Het- SL-1-18- can promote the degradation of ERα via the ubiquitin-proteasome pathway and should be further developed as a therapeutic option for ER+ breast cancer.

ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer.

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR-ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa.

Synthesis and evaluation of the diarylthiourea analogs as novel anti-cancer agents.

Ten p-nitrodiarylthiourea analogs were designed, synthesized and evaluated in breast (MCF-7, T-47D, MDA-MB-453) and prostate (DU-145, PC-3, LNCaP) cancer cell lines for their anticancer activities. The majority of the compounds were able to inhibit the growth of these six cancer cell lines at low micromolar concentrations. Compound 7 was found to be the most potent anticancer agent in this series with GI50 values of 3.16µM for MCF-7, 2.53µM for T-47D, 4.77µM for MDA-MB-453 breast cancer lines and 3.54µM for LNCaP prostate cancer cell line. These GI50 values were comparable to the parent compound, SHetA2.

Acute and chronic cadmium exposure promotes E-cadherin degradation in MCF7 breast cancer cells.

Cadmium is an environmental carcinogen that usually enters the body at minute concentrations through diet or cigarette smoke and bioaccumulates in soft tissues. In past studies, cadmium has been shown to contribute to the development of more aggressive cancer phenotypes including increased cell migration and invasion. This study aims to determine if cadmium exposure-both acute and chronic-contributes to breast cancer progression by interfering with the normal functional relationship between E-cadherin and ß-catenin. An MCF7 breast cancer cell line (MCF7-Cd) chronically exposed to 10(-7) M CdCl2 was previously developed and used as a model system to study chronic exposures, whereas parental MCF7 cells exposed to 10(-6) M CdCl2 for short periods of time were used to study acute exposures. Cadmium exposure of MCF7 cells led to the degradation of the E-cadherin protein via the ubiquitination pathway. This resulted in fewer E-cadherin/ß-catenin complexes and the relocation of active ß-catenin to the nucleus, where it interacted with transcription factor TCF-4 to modulate gene expression. Interestingly, only cells chronically exposed to cadmium showed a significant decrease in the localization of ß-catenin to the plasma membrane and an increased distance between cells. Our data suggest that cadmium exposure promotes breast cancer progression by (1) down-regulating E-cadherin, thus decreasing the number of E-cadherin/ß-catenin adhesion complexes, and (2) enhancing the nuclear translocation of ß-catenin to increase expression of cancer-promoting proteins (i.e., c-Jun and cyclin D1).

Using expression profiling to understand the effects of chronic cadmium exposure on MCF-7 breast cancer cells.

Cadmium is a metalloestrogen known to activate the estrogen receptor and promote breast cancer cell growth. Previous studies have implicated cadmium in the development of more malignant tumors; however the molecular mechanisms behind this cadmium-induced malignancy remain elusive. Using clonal cell lines derived from exposing breast cancer cells to cadmium for over 6 months (MCF-7-Cd4, -Cd6, -Cd7, -Cd8 and -Cd12), this study aims to identify gene expression signatures associated with chronic cadmium exposure. Our results demonstrate that prolonged cadmium exposure does not merely result in the deregulation of genes but actually leads to a distinctive expression profile. The genes deregulated in cadmium-exposed cells are involved in multiple biological processes (i.e. cell growth, apoptosis, etc.) and molecular functions (i.e. cadmium/metal ion binding, transcription factor activity, etc.). Hierarchical clustering demonstrates that the five clonal cadmium cell lines share a common gene expression signature of breast cancer associated genes, clearly differentiating control cells from cadmium exposed cells. The results presented in this study offer insights into the cellular and molecular impacts of cadmium on breast cancer and emphasize the importance of studying chronic cadmium exposure as one possible mechanism of promoting breast cancer progression.

Chronic cadmium exposure stimulates SDF-1 expression in an ERα dependent manner.

Cadmium is an omnipotent environmental contaminant associated with the development of breast cancer. Studies suggest that cadmium functions as an endocrine disruptor, mimicking the actions of estrogen in breast cancer cells and activating the receptor to promote cell growth. Although acute cadmium exposure is known to promote estrogen receptor-mediated gene expression associated with growth, the consequence of chronic cadmium exposure is unclear. Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims to investigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell line chronically exposed to 10(-7) M CdCl2 serves as our model system. Data suggest that prolonged cadmium exposures result in the development of more aggressive cancer phenotypes - increased cell growth, migration and invasion. The results from this study show for the first time that chronic cadmium exposure stimulates the expression of SDF-1 by altering the molecular interactions between ERα, c-jun and c-fos. This study provides a mechanistic link between chronic cadmium exposure and ERα and demonstrates that prolonged, low-level cadmium exposure contributes to breast cancer progression.

The role of cadmium and nickel in estrogen receptor signaling and breast cancer: metalloestrogens or not?

During the past half-century, incidences of breast cancer have increased globally. Various factors--genetic and environmental--have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, low-level exposure to certain heavy metals (i.e., cadmium and nickel) can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role in breast cancer development by acting as metalloestrogens--metals that bind to estrogen receptors and mimic the actions of estrogen. Since the lifetime exposure to estrogen is a well-established risk factor for breast cancer, anything that mimics its activity will likely contribute to the etiology of the disease. However, heavy metals, depending on their concentration, are capable of binding to a variety of proteins and may exert their toxicities by disrupting multiple cellular functions, complicating the analysis of whether heavy metal-induced carcinogenesis is mediated by the estrogen receptor. The purpose of this review is to discuss the various epidemiological, in vivo, and in vitro studies that show a link between the heavy metals, cadmium and nickel, and breast cancer development. We will particularly focus on the studies that test whether these two metals act as metalloestrogens in order to assess the strength of the data supporting this hypothesis.

Glycosylation of human cyclooxygenase-2 (COX-2) decreases the efficacy of certain COX-2 inhibitors.

Prostanoids play an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. The rate-limiting step in the prostanoid biosynthesis pathway is catalyzed by cyclooxygenase-2 (COX-2). COX-2 exists as two glycoforms, 72 and 74 kDa, the latter resulting from an additional glycosylation at Asn(580). In this study, Asn(580) was mutated, and the mutant and wild-type COX-2 genes were expressed in COS-1 cells to determine how glycosylation affects the inhibition of COX-2 activity by aspirin, flurbiprofen, ibuprofen, celecoxib, and etoricoxib. Results indicate that certain inhibitors were 2-5 times more effective at inhibiting COX-2 activity when the glycosylation site was eliminated, indicating that glycosylation of COX-2 at Asn(580) decreases the efficacy of some inhibitors.

Estrogen receptor regulates E2F1 expression to mediate tamoxifen resistance.

Antiestrogen resistance often develops with prolonged exposure to hormone therapies, including tamoxifen, and is a major problem in the treatment of breast cancer. Understanding the mechanisms involved in the development of antiestrogen resistance is an important step in the development of new targeted therapies. Two forms of antiestrogen resistance exist: de novo resistance and acquired resistance. To mimic acquired resistance, we have established a tamoxifen-resistant breast cancer cell line (MCF-7TamR) by treating parental MCF-7 cells with tamoxifen over a period of 6 months to select for cells with the resistant phenotype. Characterization of the MCF-7TamR cells under normal, hormone-deprived, and tamoxifen-treated conditions suggests that these cells continue to grow in the presence of tamoxifen. Additionally, a greater percentage of resistant cells enter the S phase under tamoxifen conditions, compared with parental MCF-7 cells. Consistent with these growth results, molecular analysis indicates that tamoxifen-resistant cells express higher levels of cyclin E1, cdk2, ACTR, and E2F1. Faslodex or ICI 182, 780 (ICI)-mediated degradation of estrogen receptor (ER) reduced the proliferation of these cells, as well as the level of E2F1 expression in tamoxifen-resistant cells, suggesting that tamoxifen resistance and E2F1 expression are in part dependent on ER. We further showed that tamoxifen enhances the ERalpha/Sp-1 interaction and promotes the recruitment of ERalpha and Sp-1 to the proximal promoter of E2F1 in resistant cells. Collectively, our findings suggest that tamoxifen resistance is a result of increased ERalpha/Sp-1 interaction, which enhances the expression of E2F1 to promote tamoxifen resistance.

Cadmium promotes breast cancer cell proliferation by potentiating the interaction between ERalpha and c-Jun.

Cadmium is an environmental contaminant that enters the body through diet or cigarette smoke. It affects multiple cellular processes, including cell proliferation, differentiation, and apoptosis. Recently, cadmium has been shown to function as an endocrine disruptor, to stimulate estrogen receptor alpha (ERalpha) activity and promote uterine and mammary gland growth in mice. Although cadmium exposure has been associated with the development of breast cancer, the mechanism of action of cadmium remains unclear. To address this deficit, we examined the effects of cadmium treatment on breast cancer cells. We found that ERalpha is required for both cadmium-induced cell growth and modulation of gene expression. We also determined that ERalpha translocates to the nucleus in response to cadmium exposure. Additionally, we provide evidence that cadmium potentiates the interaction between ERalpha and c-Jun and enhances recruitment of this transcription factor complex to the proximal promoters of cyclin D1 and c-myc, thus increasing their expression. This study provides a mechanistic link between cadmium exposure and ERalpha and demonstrates that cadmium plays an important role in the promotion of breast cancer.

Proto-oncogene ACTR/AIB1 promotes cancer cell invasion by up-regulating specific matrix metalloproteinase expression.

Overexpression of ACTR/AIB1 is frequently found in different cancers with distant metastasis. To address its possible involvement in tumor metastasis, we performed invasion assays to examine the effect of ACTR alteration on the invasiveness of breast cancer cells (MDA-MB-231 or T-47D) and found that high levels of ACTR are required for their strong invasiveness. Molecular analysis indicates that ACTR functions as a coactivator of AP-1 to up-regulate the expression of matrix metalloproteinases such as MMP-7 and MMP-10 and reduce cell adhesion to specific extracellular matrix proteins. These novel findings provide a mechanistic link between ACTR and MMPs, and suggest that ACTR may also play an important role in cancer progression by facilitating tumor invasion.

Direct control of cell cycle gene expression by proto-oncogene product ACTR, and its autoregulation underlies its transforming activity.

ACTR (also called AIB1 and SRC-3) was identified as a coactivator for nuclear receptors and is linked to multiple types of human cancer due to its frequent overexpression. However, the molecular mechanism of ACTR oncogenicity and its function independent of nuclear receptors remain to be defined. We demonstrate here that ACTR is required for both normal and malignant human cells to effectively enter S phase. RNA interference-mediated depletion and chromatin immunoprecipitation assays show that endogenous ACTR directly controls the expression of genes important for initiation of DNA replication, which include cdc6, cdc25A, MCM7, cyclin E, and Cdk2. Moreover, consistent with its critical role in cell cycle control, ACTR expression appears to be cell cycle regulated, which involves E2F. Interestingly, ACTR is recruited to its own promoter at the G1/S transition and activates its own expression, suggesting a positive feedback mechanism for ACTR action in the control of cell cycle progression and for its aberrant expression in cancers. Importantly, overexpression of ACTR alone transforms human mammary epithelial cells, which requires its association with E2F. These findings reveal a novel role for ACTR in cell cycle control and support the notion that the ability of aberrant ACTR to deregulate the cell cycle through E2F underlies its oncogenicity in human cancers.

ACTR/AIB1 functions as an E2F1 coactivator to promote breast cancer cell proliferation and antiestrogen resistance.

Overexpression or amplification of ACTR (also named AIB1, RAC3, p/CIP, TRAM-1, and SRC-3), a member of the p160 family of coactivators for nuclear hormone receptors, has been frequently detected in multiple types of human tumors, including breast cancer. However, its role in cancer cell proliferation and the underlying mechanism are unclear. Here, we show that overexpression of ACTR not only enhances estrogen-stimulated cell proliferation but also, more strikingly, completely negates the cell cycle arrest effect by tamoxifen and pure antiestrogens. Unexpectedly, we found that ACTR directly interacts, through its N-terminal domain, with E2F1 and is recruited to E2F target gene promoters. Elevation of ACTR in quiescent cells strongly stimulates the transcription of a subset of E2F-responsive genes that are associated with the G(1)/S transition. We also demonstrated, by adenovirus vector-mediated RNA interference, that ACTR is required for E2F1-mediated gene expression and the proliferation of estrogen receptor (ER)-negative breast cancer cells. Moreover, the ability of elevated ACTR to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between ACTR and E2F1, but not ER. Thus, our results reveal an essential role of ACTR in control of breast cancer cell proliferation and implicate the ACTR-E2F1 pathway as a novel mechanism in antiestrogen resistance.

Interleukin-8 confers androgen-independent growth and migration of LNCaP: differential effects of tyrosine kinases Src and FAK.

Interleukin-8 (IL-8), a chemokine implicated in the metastasis and angiogenesis of a variety of cancers, has been reported to be overexpressed in prostate cancer. In this study, we ascribe a new role for IL-8 in prostate cancer progression using LNCaP cells. We demonstrate that IL-8 activates the androgen receptor and confers androgen-independent growth, while serving as a potent chemotactic factor. Our evaluation of the possible signal pathways involved in androgen-independence and cell migration shows that the tyrosine kinases Src and FAK (focal adhesion kinase) are involved in IL-8-induced signaling. Pharmacological and genetic inhibitors of Src and FAK interfere with IL-8-induced cell migration, while only the Src inhibitor was able to repress androgen-independent growth. This suggests that both growth and migration depend on the activity of Src, whereas cell migration also requires the activation of FAK. Our evidence that IL-8-induced androgen-independent growth is, at least in part, due to androgen receptor activation includes (1) an inhibitor of androgen receptor activity diminishes cell growth; (2) androgen receptor transactivation potential is augmented by IL-8 and (3) androgen receptor is recruited to the promoter of prostate specific antigen (PSA) upon IL-8 treatment, based on chromatin immunoprecipitation experiments. Taken together, our data suggest that in addition to its role in metastasis and angiogenesis, IL-8 may also serve as a facilitator for androgen-independent transition of prostate cancers. To our knowledge, this is the first report about the tyrosine kinase signals and androgen receptor activation induced by IL-8 in prostate cancer cells. The observation that IL-8 mediates its growth and chemotactic effects via Src and FAK suggests the potential use for tyrosine kinase inhibitors at early stage of prostate cancer development.