Heather Beckwith

Minireview: Were the IGF Signaling Inhibitors All Bad?

Preclinical studies in the 1980s defined a role for IGF signaling in the development and sustainability of the malignant process. Subsequently, antibody, tyrosine kinase, and ligand inhibitors of the IGF receptor were manufactured. In the past decade, numerous clinical trials have tested the efficacy of IGF receptor inhibitors in the treatment of advanced tumors. Early-phase trials in heavily pretreated populations showed promise with complete or partial responses in a few patients and stable disease in many more. Unfortunately, the results of the early-phase trials did not pan out to later-phase trials. The lack of use of biomarkers to define subsets of patients that may benefit from IGF receptor blockade and compensatory signaling via other growth factor receptors such as the insulin, GH, and epidermal growth factor receptors may have played a role in the lack of efficacy of IGF receptor inhibition in phase III trials. Although these trials failed to show benefit, the trials have revealed previously unknown knowledge regarding the complex nature of IGF signaling. The knowledge obtained from these trials will be useful in designing future trials studying inhibitors of growth factor signaling.

Insulin-like growth factors, insulin, and growth hormone signaling in breast cancer: implications for targeted therapy.

OBJECTIVE In recent decades, multiple therapeutics targeting the estrogen and human epidermal growth factor-2 (HER2) receptors have been approved for the treatment of breast cancer. METHODS This review discusses a number of growth factor pathways that have been implicated in resistance to both anti-estrogen and HER2-targeted therapies. The association between growth factors and breast cancer is well established. Over decades, numerous laboratories have studied the link between insulin-like growth factor (IGF), insulin, and growth hormone (GH) to the development and progression of breast cancer. RESULTS Although preclinical data demonstrates that blockade of these receptors inhibits breast cancer growth, progression, and drug resistance, therapies targeting the IGF, insulin, and GH receptors (GHRs) have not been successful in producing significant increases in progression-free, disease-free, or overall survival for patients with breast cancer. The failure to demonstrate a benefit of growth factor blockade in clinical trials can be attributed to redundancy in IGF, insulin, and GHR signaling pathways. All 3 receptors are able to activate oncogenic phosphoinositide-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. CONCLUSION Consequently, multitargeted blockade of growth factor receptors and their common downstream kinases will be necessary for the successful treatment of breast cancer.

Relative effectiveness of adjuvant chemotherapy for invasive lobular compared with invasive ductal carcinoma of the breast

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) have distinct clinical, pathologic, and genomic characteristics. The objective of the current study was to compare the relative impact of adjuvant chemotherapy on the survival of patients with ILC versus those with IDC.

Acquired Tamoxifen Resistance in MCF-7 Breast Cancer Cells Requires Hyperactivation of eIF4F-Mediated Translation

While selective estrogen receptor modulators, such as tamoxifen, have contributed to increased survival in patients with hormone receptor-positive breast cancer, the development of resistance to these therapies has led to the need to investigate other targetable pathways involved in oncogenic signaling. Approval of the mTOR inhibitor everolimus in the therapy of secondary endocrine resistance demonstrates the validity of this approach. Importantly, mTOR activation regulates eukaryotic messenger RNA translation. Eukaryotic translation initiation factor 4E (eIF4E), a component of the cap-dependent translation complex eIF4F, confers resistance to drug-induced apoptosis when overexpressed in multiple cell types. The eIF4F complex is downstream of multiple oncogenic pathways, including mTOR, making it an appealing drug target. Here, we show that the eIF4F translation pathway was hyperactive in tamoxifen-resistant (TamR) MCF-7L breast cancer cells. While overexpression of eIF4E was not sufficient to confer resistance to tamoxifen in MCF-7L cells, its function was necessary to maintain resistance in TamR cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of TamR cells and partially restored sensitivity to tamoxifen. Further, the use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, the use of a pharmacologic agent which inhibited the eIF4E-eIF4G interaction also decreased the proliferation and anchorage-dependent colony formation in TamR cells. These results highlight the eIF4F complex as a promising target for patients with acquired resistance to tamoxifen and, potentially, other endocrine therapies.

Distribution of 21-Gene Recurrence Scores Among Breast Cancer Histologic Subtypes

CONTEXT - The 21-gene recurrence score (RS) provides a probability of distant recurrence for estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers. The utility of RS for rarer histologic subtypes of breast cancer is uncertain. OBJECTIVE - To determine the distribution of RS among various histologic subtypes using a population database. DESIGN - Women between the ages of 18 and 75 with estrogen receptor-positive, HER2-negative breast cancer and known RS results were identified using the Surveillance, Epidemiology, and End Results database. Recurrence scores were categorized into risk groups using both traditional and Trial Assigning Individualized Options for Treatment cutoffs. Multivariable logistic regression was used to determine factors associated with high-risk RS. RESULTS - We identified 45 618 patients with stage I to III, estrogen receptor-positive, HER2-negative breast cancer who had RS available. Overall, 3087 (7%) and 6337 (14%) of cancers were classified as high risk based on traditional and Trial Assigning Individualized Options for Treatment RS cutoffs, respectively. The proportion of high-risk RS ranged from 1% (tubular, 2 of 225) to 68% (medullary, 13 of 19) and 4% (tubular, 10 of 225) to 79% (medullary, 15 of 19) for traditional and Trial Assigning Individualized Options for Treatment cutoffs, respectively. Based on multivariable logistic regression (excluding medullary), subtypes other than invasive ductal carcinoma and papillary carcinoma were significantly associated with lower RS. The strongest predictors of a high-risk RS were higher tumor grade and negative progesterone receptor status. CONCLUSIONS - We identified distinct distributions of RS among different histologic subtypes of breast cancer. Excluding medullary carcinoma, histologic subtypes other than invasive ductal carcinoma and papillary carcinoma all predict lower RS.

Abstract S5-07: Aromatase inhibitors and endothelial function: Is there an association with early cardiovascular disease?

Background: As more women are cured from their breast cancer, survivors with early stage breast cancer are at greater risk of dying from cardiovascular disease than their breast cancer. Aromatase inhibitors (AI) have been shown to reduce breast cancer-related mortality in women with estrogen receptor (ER)-positive disease which makes up 75% of all breast cancer cases. The use of AIs has been associated with higher rates of hypertension, hypercholesterolemia, angina pectoris and ischemic cardiovascular disease. In the aging population taking AIs, little is known about the direct impact of AIs on endothelial function, a predictor of cardiovascular disease. Endothelial dysfunction identified by reactive hyperemia using Endo-PAT has been associated with an increased risk of cardiac adverse events, independent of Framingham risk score. Methods: At the University of Minnesota in 2014-2015, 25 healthy postmenopausal women and 36 postmenopausal women with locally advanced breast cancer and prescribed an aromatase inhibitor were identified. Subjects with a history of hypertension or hyperlipidemia were excluded. Consented subjects underwent biomarker analysis and pulse wave analysis using the HDI/Pulse Wave CR-2000 Cardiovascular Profiling System and pulse contour analysis using the Endo-PAT2000 system. Biomarkers and functional test markers were compared between cases and controls using T-tests and Wilcoxon Rank-Sum tests. Results: Mean age (61.7 vs 58.8 years), body mass index (27.4 vs 26.2 kg/m2), race (93% vs 92% Caucasian), and tobacco use (100% nonsmokers) were similar between cases and controls, respectively. Mean systolic blood pressure (BP) was elevated in cases (128.3 mmHg vs 114.5 mmHg, p=0.0006). There were no differences in lipid profiles. Median ultrasensitive estradiol levels were reduced in cases (2 vs 15 pg/mL, p Conclusion: Postmenopausal women with breast cancer on AIs have reductions in endothelial function, a predictor of adverse cardiovascular disease (acute coronary syndrome, chest pain, myocardial infarction, cardiac death). With the growing trend that longer duration of endocrine therapy is needed, further work is needed to confirm these findings. Citation Format: Blaes AH, Beckwith H, Hebbel R, Solovey A, Potter D, Yee D, Vogel R, Luepker R, Duprez D. Aromatase inhibitors and endothelial function: Is there an association with early cardiovascular disease? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S5-07.

Abstract P4-06-06: Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells

Targeting endocrine resistant breast cancer cells has been successfully accomplished by the use of mTORC1 inhibitors in combination with either aromatase inhibitors or tamoxifen. While there was preclinical data suggesting that targeting of the type I IGF receptor (IGF1R) might also be a target for endocrine resistant cells, clinical trials using IGF1R monoclonal antibodies showed no advantage and even suggested potential harm for the combination. IGF1R inhibition results in the disruption of an endocrine feedback loop and results in elevation of the pituitary growth factor growth hormone (GH). GH is involved in normal human growth, development, metabolism, and longevity. Growth hormone released from the pituitary stimulates production of IGF-I from the liver. The GH/IGF-1 axis is known to promote mammary gland hyperplasia and breast cancer carcinogenesis. Previous studies have shown that GH promotes carcinogenesis independently of insulin-like growth factor. The role of GH in specific subtypes of breast carcinoma remains to be defined. Our laboratory has found that estrogen receptor positive breast cancer cell lines up regulated oncogenic STAT5, Akt, and MAPK signaling pathways in response to GH. Tamoxifen resistant (TamR) and long-term estrogen deprived (LTED) breast cancer cell lines derived from MCF-7 and T47D cells demonstrated increased oncogenic signaling in response to GH compared to the corresponding parental cell lines from which they were derived. In contrast, GH stimulation of a triple negative breast cancer cell line (MDA-MB-231) failed to induce signaling via these oncogenic pathways. Although GH treatment of parental, TamR, and LTED breast cancer cells had minimal effect on cell proliferation, cell cycle progression, or migration; GH signaling protected cells from cytotoxic chemotherapy induced apoptosis. This data shows that GH signaling is found in endocrine sensitive and resistant cells. Furthermore, GH may function in protecting cells from cell death induced by either endocrine or cytotoxic drugs. GH receptor blockade may be a valid treatment option for endocrine resistant breast cancer. Citation Format: Heather C Beckwith, Douglas Yee. Growth hormone induction of oncogenic signaling promotes survival of endocrine resistant breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-06.

Abstract IA17: Targeting downstream effectors of growth factor signaling

Transmembrane growth factor receptors mediate signaling through multiple intracellular pathways. In breast cancer cells, the type I insulin-like growth factor receptor (IGF1R) has been implicated in the malignant phenotype. However, clinical trials with anti-IGF1R antibodies have been disappointing, in part, due to adaptive feedback pathways stimulated when IGF1R is blocked. To determine whether IGF1R inhibition could be enhanced by disrupting other pathways, we evaluated gene expression induced by receptor activation. In previous work, we found that xCT (SLC7A11) mRNA expression was increased by IGF-I in estrogen receptor (ER) positive breast cancer cell lines (MCF-7, T47D, and ZR-75-1) in an insulin receptor substrate-1 (IRS-1) dependent manner. xCT encodes the functional subunit of the heterodimeric plasma membrane transport system xC- critical for the cellular uptake of cystine to generate glutathione to modulate cellular redox control. IGF-I increased xC- transporter expression and function to control cellular redox levels. In MCF-7 cells, IGF-I-stimulated monolayer and anchorage-independent growth was suppressed by infecting cells with xCT shRNA or by treating cells with the xC- chemical inhibitor sulfasalazine (SASP). Anchorage-independent growth assays showed that disruption of xC- function by SASP sensitized cellular response of MCF-7 cells to anti-IGF-IR inhibitors (monoclonal antibody huEM164 and tyrosine kinase inhibitor NVP-AEW-541). IGF1R also activates PI3K/Akt/mTORC1 signaling to affect ER phosphorylation and mRNA cap dependent translation. In tamoxifen resistant cells, IGF1R is lost yet PI3K signaling is maintained. Since mRNA cap dependent translation is increased by PI3K signaling, we determined if inhibition of the eIF4F translation pathway would affect endocrine responsive and tamoxifen resistant cells. The eIF4F translation pathway is activated by IGF1R in wild-type cells and hyperactive in tamoxifen resistant MCF-7L (TamR) breast cancer cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of parental and TamR cells. Use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, pharmacologic inhibition of the eIF4E-eIF4G interaction also decreased the proliferation and anchorage dependent colony formation in TamR cells. Taken together, these data show that IGF1R activation stimulates multiple downstream effectors important for breast cancer cell biology. Inhibition of selected downstream signaling molecules is likely to have synergy with anti-IGF-IR drugs. Citation Format: Yuzhe Yang, Dedra Fagan, Lynsey Fettig Anderson, Kelly LaPara, Xihong Zhang, Aleksandra Ochnik, Jie Ying Chan, Heather Beckwith, Douglas Yee. Targeting downstream effectors of growth factor signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA17.

Abstract P4-07-03: Rapamycin and Dalotuzumab in combination inhibit parental and endocrine resistant breast cancer cells.

The PI3K/Akt/mTOR pathway is involved in breast cancer resistance to endocrine therapy. Inhibitors of mTOR have been shown to have benefit for patients with ER positive tumors that have developed endocrine therapy resistance. Inhibition of mTOR triggers a negative feedback loop resulting in enhanced phosphorylation of Akt and subsequent breast cancer cell proliferation via the IGF signaling pathway. Therefore, blockade of both IGF-1R and mTOR may be necessary to completely suppress this pathway. Our laboratory studied the effect of dual inhibition of mTOR and IGF-1R with rapamycin and dalotuzumab respectively on parental and endocrine resistant MCF-7 breast cancer cell tumorigenesis. Immunoblotting demonstrates parental MCF-7L cells treated with rapamycin alone demonstrate increased phosphorylation of Akt. Inhibition of phosphorylation of Akt can be achieved with combined treatment with rapamycin and dalotuzumab at concentrations of 5 nM and 2 µg/mL respectively. In addition to inhibition of pAkt, combined treatment inhibits phosphorylation of S6K1 and the translational repressor protein, 4eBP1. Furthermore, MTT proliferation assay and soft agar assay demonstrate inhibition of parental cell proliferation and colonization respectively with combined rapamycin and dalotuzumab treatment. In order to examine the effect of combination therapy in endocrine resistant cell lines, we established tamoxifen resistant (TamR) and long term estrogen deprived (LTED) cell lines. TamR cells were generated by culturing MCF-7L cells in the presence of tamoxifen for more than 1 year. LTED cells were generated by culturing MCF-7L cells in the absence of estrogen for more than 6 months. Proliferation of TamR and LTED cells treated with rapamycin at a concentration of 5nM and dalotuzumab at a concentration of 2µg/mL in combination was significantly inhibited. We conclude that combination of IGF1R and mTOR inhibition might be necessary to inhibit endocrine resistant breast cancers. Ongoing clinical trials will test this combination of drugs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-03.