Jeff S. Jasper

27-Hydroxycholesterol Links Hypercholesterolemia and Breast Cancer Pathophysiology

Cholesterol and Cancer Obesity and high cholesterol levels are associated with an increased risk of breast cancer in post-menopausal women. Nelson et al. (p. 1094) found that a specific metabolite of cholesterol, 27-hydroxycholesterol (27HC), promoted tumor growth and metastasis in mouse models of mammary cancer by serving as a partial agonist for the estrogen receptor and the liver X receptor. The most aggressive human breast cancers were found to express the highest level of the enzyme that converts cholesterol to 27HC, suggesting that 27HC produced within tumors (in addition to circulating 27HC) may contribute to tumorigenesis. The activity of a specific metabolite of cholesterol may help explain why obesity is a risk factor for breast cancer. Hypercholesterolemia is a risk factor for estrogen receptor (ER)–positive breast cancers and is associated with a decreased response of tumors to endocrine therapies. Here, we show that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol and an ER and liver X receptor (LXR) ligand, increases ER-dependent growth and LXR-dependent metastasis in mouse models of breast cancer. The effects of cholesterol on tumor pathology required its conversion to 27HC by the cytochrome P450 oxidase CYP27A1 and were attenuated by treatment with CYP27A1 inhibitors. In human breast cancer specimens, CYP27A1 expression levels correlated with tumor grade. In high-grade tumors, both tumor cells and tumor-associated macrophages exhibited high expression levels of the enzyme. Thus, lowering circulating cholesterol levels or interfering with its conversion to 27HC may be a useful strategy to prevent and/or treat breast cancer.

Systematic identification of signaling pathways with potential to confer anticancer drug resistance.

Pathway-centric screening reveals new mechanisms of drug resistance and combination therapeutic strategies. Finding New Targets for Drug-Resistant Cancers The development of drug resistance is a common problem in cancer patients. Knowing how drug resistance emerged in a tumor can inform clinical strategy. Martz et al. devised a drug screen to identify pathways of resistance when cancer cells were treated with drugs that are used in the clinic. Along with pathways known to mediate drug resistance, such as the MAPK and PI3K pathways, activation of the Notch1 pathway caused drug resistance in various types of cancer cells in culture. Inhibiting Notch1 signaling restored drug efficacy in cells in culture and in xenografts in mice. Intriguingly, Notch signaling mediated drug resistance to an estrogen receptor–targeted therapy used in breast cancer and to a kinase-targeted therapy used in melanoma, suggesting that this single pathway may be important in multiple types of drug-resistant cancers. Indeed, tumors of some patients with relapsed breast cancer or melanoma had increased markers of Notch1 signaling. In the Research Article by Winter et al. also in this issue, this screening method identified a pathway of drug resistance in bone marrow cancer. More generally, by screening entire signaling pathways instead of individual genes, the work of Martz et al. shows how we can quickly map pathways to the diverse properties of cancer cells. Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)–mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAFV600E melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Lysine-specific histone demethylase 1 inhibitors control breast cancer proliferation in ERα-dependent and independent manners

Lysine specific demethylase 1 (LSD1, also known as KDM1) is a histone modifying enzyme that regulates the expression of many genes important in cancer progression and proliferation. It is present in various transcriptional complexes including those containing the estrogen receptor (ER). Indeed, inhibition of LSD1 activity and or expression has been shown to attenuate estrogen signaling in breast cancer cells in vitro, implicating this protein in the pathogenesis of cancer. Herein we describe experiments that utilize small molecule inhibitors, phenylcyclopropylamines, along with small interfering RNA to probe the role of LSD1 in breast cancer proliferation and in estrogen-dependent gene transcription. Surprisingly, whereas we have confirmed that inhibition of LSD1 strongly inhibits proliferation of breast cancer cells, we have determined that the cytostatic actions of LSD1 inhibition are not impacted by ER status. These data suggest that LSD1 may be a useful therapeutic target in several types of breast cancer; most notably, inhibitors of LSD1 may have utility in the treatment of ER-negative cancers for which there are minimal therapeutic options.

Obesity, Cholesterol Metabolism, and Breast Cancer Pathogenesis

Obesity and altered lipid metabolism are risk factors for breast cancer in pre- and post-menopausal women. These pathologic relationships have been attributed in part to the impact of cholesterol on the biophysical properties of cell membranes and to the influence of these changes on signaling events initiated at the membrane. However, more recent studies have indicated that the oxysterol 27-hydroxycholesterol (27HC), and not cholesterol per se, may be the primary biochemical link between lipid metabolism and cancer. The enzyme responsible for production of 27HC from cholesterol, CYP27A1, is expressed primarily in the liver and in macrophages. In addition, significantly elevated expression of this enzyme within breast tumors has also been observed. It is believed that 27HC, acting through the liver X receptor in macrophages and possibly other cells, is involved in maintaining organismal cholesterol homeostasis. It has also been shown recently that 27HC is an estrogen receptor agonist in breast cancer cells and that it stimulates the growth and metastasis of tumors in several models of breast cancer. These findings provide the rationale for the clinical evaluation of pharmaceutical approaches that interfere with cholesterol/27HC synthesis as a means to mitigate the impact of cholesterol on breast cancer pathogenesis. Cancer Res; 74(18); 4976-82. ©2014 AACR.

Delineation of a FOXA1/ERα/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer

Tamoxifen, a selective estrogen receptor (ER) modulator (SERM), remains a frontline clinical therapy for patients with ERα-positive breast cancer. However, the relatively rapid development of resistance to this drug in the metastatic setting remains an impediment to a durable response. Although drug resistance likely arises by many different mechanisms, the consensus is that most of the implicated pathways facilitate the outgrowth of a subpopulation of cancer cells that can either recognize tamoxifen as an agonist or bypass the regulatory control of ERα. Notable in this regard is the observation here and in other studies that expression of anterior gradient homology 2 (AGR2), a known proto-oncogene and disulfide isomerase, was induced by both estrogen (17β-estradiol, E2) and 4-hydroxytamoxifen (4OHT) in breast cancer cells. The importance of AGR2 expression is highlighted here by the observation that (i) its knockdown inhibited the growth of both tamoxifen-sensitive and -resistant breast cancer cells and (ii) its increased expression enhanced the growth of ERα-positive tumors in vivo and increased the migratory capacity of breast cancer cells in vitro. Interestingly, as with most ERα target genes, the expression of AGR2 in all breast cancer cells examined requires the transcription factor FOXA1. However, in tamoxifen-resistant cells, the expression of AGR2 occurs in a constitutive manner, requiring FOXA1, but loses its dependence on ER. Taken together, these data define the importance of AGR2 in breast cancer cell growth and highlight a mechanism where changes in FOXA1 activity obviate the need for ER in the regulation of this gene. Implications: These findings reveal the transcriptional interplay between FOXA1 and ERα in controlling AGR2 during the transition from therapy-sensitive to -resistant breast cancer and implicate AGR2 as a relevant therapeutic target. Mol Cancer Res; 12(12); 1829–39. ©2014 AACR.

CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662-73. ©2017 AACR.

ELF3 is a repressor of androgen receptor action in prostate cancer cells.

The androgen receptor (AR) has a critical role in the development and progression of prostate cancer (PC) and is a major therapeutic target in this disease. The transcriptional activity of AR is modulated by the coregulators with which it interacts, and consequently deregulation of cofactor expression and/or activity impacts the expression of genes whose products can have a role in PC pathogenesis. Here we report that E74-like factor 3 (ELF3), a member of the ETS family of transcription factors, is a repressor of AR transcriptional activity. Exogenous expression of ELF3 represses AR transcriptional activity when assessed using reporter-based transfection assays or when evaluated on endogenous AR target genes. Conversely, ELF3 knock down increases the AR transcriptional activity. Biochemical dissection of this activity indicates that it results from the physical interaction between ELF3 and AR and that this interaction inhibits the recruitment of AR to specific androgen response elements within target gene promoters. Significantly, we observed that depletion of ELF3 expression in LNCaP cells promotes cell migration, whereas increased ELF3 expression severely inhibits tumor growth in vitro and in a mouse xenograft model. Taken together, these results suggest that modulation of ELF3 expression and/or AR/ELF3 interaction may have utility in the treatment of PC.

MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents a major challenge in the clinic due to its lack of reliable prognostic markers and targeted therapies. Accumulating evidence strongly supports the notion that microRNAs (miRNAs) are involved in tumorigenesis and could serve as biomarkers for diagnostic purposes. To identify miRNAs that functionally suppress metastasis of TNBC, we employed a concerted approach with selecting miRNAs that display differential expression profiles from bioinformatic analyses of breast cancer patient databases and validating top candidates with functional assays using breast cancer cell lines and mouse models. We have found that miR-148a exhibits properties as a tumor suppressor as its expression is inversely correlated with the ability of both human and mouse breast cancer cells to colonize the lung in mouse xenograft tumor models. Mechanistically, miR-148a appears to suppress the extravasation process of cancer cells, likely by targeting two genes WNT1 and NRP1 in a cell non-autonomous manner. Importantly, lower expression of miR-148a is detected in higher-grade tumor samples and correlated with increased likelihood to develop metastases and poor prognosis in subsets of breast cancer patients, particularly those with TNBC. Thus, miR-148a is functionally defined as a suppressor of breast cancer metastasis and may serve as a prognostic biomarker for this disease.

Effect of aerobic training on the host systemic milieu in patients with solid tumours: an exploratory correlative study.

Background:Few studies have investigated the effects of exercise on modulation of host factors in cancer patients. We investigated the efficacy of chronic aerobic training on multiple host-related effector pathways in patients with solid tumours.Patients and Methods:Paired peripheral blood samples were obtained from 44 patients with solid tumours receiving cytotoxic therapy and synthetic erythropoietin (usual care; n=21) or usual care plus supervised aerobic training (n=23) for 12 weeks. Samples were characterised for changes in immune, cytokine and angiogenic factors, and metabolic intermediates. Aerobic training consisted of three supervised cycle ergometry sessions per week at 60% to 100% of peak oxygen consumption (VO2peak), 30–45 min per session, for 12 weeks following a nonlinear prescription.Results:The between-group delta change in cardiopulmonary function was +4.1 ml kg −1 min−1, favouring aerobic training (P<0.05). Significant pre–post between-group differences for five cytokine and angiogenic factors (HGF, IL-4, macrophage inflammatory protein-1β (MIP-1β), vascular endothelial growth factor (VEGF), and TNF-α) also favour the aerobic training group (P’s<0.05). These reductions occurred in conjunction with nonsignificant group differences for T lymphocytes CD4+, CD8+, and CD8+/CD45RA (P<0.10). For these factors, circulating concentrations generally increased from baseline to week 12 in the aerobic training group compared with decreases or no change in the usual care group. No significant changes in any metabolic intermediates were observed.Conclusions:Aerobic training alters host availability of select immune–inflammatory effectors in patients with solid tumours; larger confirmatory studies in more homogenous samples are warranted.

ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

ABL kinase inhibitors could prevent breast cancer cells from breaking down bone to promote metastasis. Metastatic breast tumors break down bone Most breast cancer cells activate the breakdown of bone to promote metastases. Wang et al. found that the ABL kinase isoforms ABL1 and ABL2 enhanced the ability of breast cancer cells to invade and break down bone in mice. In breast cancer cells, the ABL kinases activated the transcriptional coactivator TAZ and the transcription factor STAT5, which triggered the transcription of genes encoding factors that activate osteoclasts (cells that break down bone) and those that enhance the survival of breast cancer cells in the bone microenvironment. An ABL-specific inhibitor decreased bone metastasis in mice, thus suggesting that blocking ABL kinase activity could prevent breast cancer cells from promoting bone metastasis. Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone.