Maeve Mullooly

The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer?

The ADAMs are transmembrane proteins implicated in proteolysis and cell adhesion. Forty gene members of the family have been identified, of which 21 are believed to be functional in humans. As proteases, their main substrates are the ectodomains of other transmembrane proteins. These substrates include precursor forms of growth factors, cytokines, growth factor receptors, cytokine receptors and several different types of adhesion molecules. Although altered expression of specific ADAMs has been implicated in different diseases, their best-documented role is in cancer formation and progression. ADAMs shown to play a role in cancer include ADAM9, ADAM10, ADAM12, ADAM15 and ADAM17. Two of the ADAMs, i.e., ADAM10 and 17 appear to promote cancer progression by releasing HER/EGFR ligands. The released ligands activate HER/EGFR signalling that culminates in increased cell proliferation, migration and survival. Consistent with a causative role in cancer, several ADAMs are emerging as potential cancer biomarkers for aiding cancer diagnosis and predicting patient outcome. Furthermore, a number of selective ADAM inhibitors, especially against ADAM10 and ADAM17, have been shown to have anti-cancer effects. At least one of these inhibitors is now undergoing clinical trials in patients with breast cancer.

ADAM-17: a novel therapeutic target for triple negative breast cancer

BACKGROUND Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.BACKGROUND Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells.

Background:Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC.Methods:We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines.Results:D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells.Conclusion:Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.

ADAM10: a new player in breast cancer progression?

Background:The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes.Methods:ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration.Results:Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer.Conclusions:Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.

Comparative antiproliferative effects of iniparib and olaparib on a panel of triple-negative and non-triple-negative breast cancer cell lines

PARP inhibitors, both as monotherapy and in combination with cytotoxic drugs, are currently undergoing clinical trials in several different cancer types. In this investigation, we compared the antiproliferative activity of two PARP/putative PARP inhibitors, i.e., olaparib and iniparib, in a panel of 14 breast cancer cell lines (seven tripe-negative and seven non-triple-negative). In almost all cell lines investigated, olaparib was a more potent inhibitor of cell growth than iniparib. Inhibition by both drugs was cell line-dependent and independent of the molecular subtype status of the cells, i.e., whether cells were triple-negative or non-triple negative. Although the primary target of PARP inhibitors is PARP1, no significant association was found between baseline levels of PARP1 activity and inhibition with either agent. Similarly, no significant correlation was evident between sensitivity and levels of CDK1, BRCA1 or miR-182. Combined addition of olaparib and either the CDK1 inhibitor, RO-3306 or a pan HER inhibitor (neratinib, afatinib) resulted in superior growth inhibition to that obtained with olaparib alone. We conclude that olaparib, in contrast to iniparib, is a strong inhibitor of breast cancer cell growth and may have efficacy in breast cancer irrespective of its molecular subtype, i.e., whether HER2-positive, estrogen receptor (ER)-positive or triple-negative. Olaparib, in combination with a selective CDK1 inhibitor or a pan HER inhibitor, is a potential new approach for treating breast cancer.

The ADAMs family of proteases as targets for the treatment of cancer

ABSTRACT The ADAMs (a disintegrin and metalloproteases) are transmembrane multidomain proteins implicated in multiple biological processes including proteolysis, cell adhesion, cell fusion, cell proliferation and cell migration. Of these varied activities, the best studied is their role in proteolysis. However, of the 22 ADAMs believed to be functional in humans, only approximately a half possess matrix metalloproteinase (MMP)-like protease activity. In contrast to MMPs which are mostly implicated in the degradation of extracellular matrix proteins, the main ADAM substrates are the ectodomains of type I and type II transmembrane proteins. These include growth factor/cytokine precursors, growth factor/cytokine receptors and adhesion proteins. Recently, several different ADAMs, especially ADAM17, have been shown to play a role in the development and progression of multiple cancer types. Consistent with this role in cancer, targeting ADAM17 with either low molecular weight inhibitors or monoclonal antibodies was shown to have anti-cancer activity in multiple preclinical systems. Although early phase clinical trials have shown no serious side effects with a dual ADAM10/17 low molecular weight inhibitor, the consequences of long-term treatment with these agents is unknown. Furthermore, efficacy in clinical trials remains to be shown.

The ADAMs: New Therapeutic Targets for Cancer?

The ADAMs are transmembrane proteins implicated in a variety of biological processes including proteolysis, cell signalling, angiogenesis, cell migration, and cell adhesion. Of the 21 ADAMs believed to be functional in humans, approximately one half have been shown to possess protease activity. As proteases, the main ADAM substrates are the ectodomains of transmembrane proteins, especially growth factor precursors, growth factor receptors, and adhesion proteins. Recently, several different ADAMs have been shown to play a role in cancer formation and progression. These include ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, and ADAM28. Consistent with a causative role in cancer, several ADAMs, especially ADAMs 10 and 17, are emerging as potential therapeutic targets for cancer treatment. Indeed, targeting these ADAMs with either low molecular weight inhibitors or monoclonal antibodies has been shown to have anticancer activity in multiple preclinical systems. Although early phase clinical trials have shown no serious side effects with a dual ADAM10/17 low molecular weight inhibitor, the consequences of long-term treatment with these agents are unknown.

Associations between obesity, smoking and lymph node status at breast cancer diagnosis in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial

Introduction There is evidence suggesting that smoking and obesity prior to a breast cancer diagnosis is associated with poorer outcomes. In this study, we investigate the associations between smoking and obesity prior to a breast cancer diagnosis and the presence of lymph node metastases at diagnosis. Methods Women with stage I-III breast cancer (n = 3,304) were identified from the National Cancer Institute’s Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. Univariable and multivariable log-binomial models were used to estimate relative risks (RR) and 95% confidence intervals (CIs) for associations between lymph node positive breast cancer and; i) smoking, and ii) obesity prior to diagnosis. Results Pre-diagnostic smoking/obesity was not associated with lymph node metastasis at diagnosis in multivariable analyses; (RR 0.82, 95%CI 0.61, 1.10) and (RR 0.95, 95% CI 0.81, 1.12), respectively. Conclusion Obesity and smoking information was recorded a number of years prior to breast cancer diagnosis, therefore these findings should to be replicated in a larger cohort of women, with more detailed smoking and obesity information.

Abstract 4235: Application of convolutional neural networks to breast biopsies to uncover tissue correlates of mammographic breast density

Background: High percent mammographic density (MD), which reflects the relative fibroglandular tissue content of the breast, is one of the strongest breast cancer risk factors; however, the pathologic mediators of this risk are unknown. We hypothesize that analysis of breast tissue sections using deep learning approaches may characterize histologic features that underpin risk associated with high MD. Methods: Non-targeted HE 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2017-4235

Abstract 4283: Relationship between mammographic breast density and measures of terminal duct lobular unit involution among women diagnosed with estrogen receptor positive breast cancer

Introduction: High mammographic breast density (MD) and reduced levels of terminal duct lobular unit (TDLU) involution (the histologic source of most breast cancers) have been associated with increased risk of developing breast cancer. Data relating MD and TDLU involution to breast cancer characteristics and outcomes are sparse. Therefore, we assessed these relationships among women with invasive ER-positive breast cancer. Methods: The analysis focused on women with ER-positive breast cancers who were diagnosed at Kaiser Permanente Northwest (1990-2008) and followed through the end of 2010. Cases comprised of those who died of breast cancer (n = 54) and controls those that did not die of breast cancer (n = 180) over similar follow-up. Three reproducible measures that are inversely related to TDLU involution were evaluated in digitized hematoxilin and eosin stained sections in benign breast tissues surrounding the tumors: TDLU counts per unit area, TDLU span and median number of acini per TDLU. Percentage MD was estimated from digitized mammograms using computer-assisted thresholding software (Cumulus). Univariate associations between TDLU measurements and patient characteristics, tumor size, and disease stage at diagnosis, were calculated using Mann Whitney Wilcoxon rank test. TDLU measurements were related to baseline MD using analysis of covariance models and adjusted for age, body mass index (BMI), tumor size, stage, year of diagnosis and smoking. Results: TDLUs were observed in 95% of cases and 89% of controls. All TDLU measurements declined with age (p 2cm) (p = 0.07 and p = 0.06, respectfully). All TDLU measures were associated with MD among controls (TDLU count: p = 0.04; TDLU span: p = 0.06; median acini count per TDLU: p = 0.01), whereas among cases only, TDLU span showed a significant association MD (p = 0.003). Conclusion: Preliminarily, our data suggest that among women with non-fatal ER-positive breast cancers, TDLU involution was associated with localized tumor stage, size and MD, whereas these relationships were less evident among women who died of their disease. Ongoing analyses will determine whether measures of MD and TDLU involution are independent predictors of breast cancer outcomes in this patient population. Citation Format: Maeve Mullooly, Sarah J. Nyante, Ruth M. Pfeiffer, Renata Cora, Jonine D. Figueroa, Robert N. Hoover, Andrew G. Glass, Erin J. Aiello Bowles, Louise A. Brinton, Amy Berrington de Gonzalez, Sherman E. Mark, Gretchen L. Gierach. Relationship between mammographic breast density and measures of terminal duct lobular unit involution among women diagnosed with estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4283.