Keith Brennan is working on Notch and Wnt signalling, both of which are required for cell-cell communication in the breast. Keith has shown that Notch signalling is dysregulated in breast cancer and that it is required to maintain the transformed phenotype of breast cancer cell lines by regulating apoptosis.

Professor of Developmental Signalling (M.A., B.A., Ph.D.)

The University of Manchester
Michael Smith Building
Oxford Road
Manchester
M13 9PT

Tel: +44 (0)161 275 5576, +44 (0)161 275 1517
Email: keith.brennan@manchester.ac.uk

Website

Wellcome Trust Centre for Cell-Matrix Research

I have always been interested in how a handful of signalling pathways can direct animal development producing many different cell types and behaviours. In particular, I have been interested in how crosstalk between signalling pathways can create greater diversity in signalling outputs and therefore cell types. During my PhD and initial postdoctoral research, I demonstrated that the Notch and Wnt pathways are intimately intertwined during embryonic and larval Drosophila development. This work showed that the Notch protein is required to suppress the expression of Wnt target genes prior to the receipt of a Wnt signal, and that a crosstalk between the two signalling pathways is required for Wnt signalling to ablate this Notch-mediate repression and initiate cell fate specification. Within my own lab, I have explored the molecular mechanism underpinning this crosstalk, demonstrating that Dishevelled, a component of the Wnt pathway, can attenuate Notch signalling by promoting the proteosomal degradation of RBP-J?/CBF1, the transcription factor at the base of the Notch pathway. In addition, my lab has shown that there is a crosstalk in the opposite direction with the Notch protein and Notch signalling disrupting signalling through the Wnt pathway.

During my second postdoctoral fellowship, I developed my second major research interest looking at how developmental signalling pathways regulate cell behaviours like proliferation, apoptosis and migration, and how the regulation of these cell behaviours by developmental signalling pathways contributes to cellular transformation and tumour initiation. This work started by looking at how oncogenic Wnt signals are detected by mammary epithelial cells within the developing murine mammary gland. During this time, I also demonstrated that the LRP5&6 proteins are the Wnt receptor components that are responsible for transducing the signal across the plasma membrane. Since establishing my own lab, I have focused on Notch signalling in mammary gland development and breast cancer, demonstrating that the Notch pathway is aberrantly activated in invasive breast cancer and a subset of ductal carcinoma in situ (a pre-invasive breast lesion). Within DCIS, Notch signalling is likely to be a useful predictive marker, as lesions with elevated Notch signalling recur, whilst lesions with little or no Notch signalling don’t. My group has also shown that Notch signalling induces a profound resistance to apoptosis in breast cancer and that inhibiting Notch sensitises breast cancer cells to chemotherapeutic agents. This work has also unravelled the underlying molecular mechanism, showing that Notch signalling activates Akt. Parallel work with Rob Clarke’s lab has found that Notch signalling is required for the self renewal and maintenance of breast cancer stem cells. Together this work is paving the way to initiating clinical trials for Notch inhibitors for the treatment of breast cancer. Finally, my group has found, very recently, that elevated EDAR signalling can also cause mammary gland tumourigenesis and they are currently investigating how this happens.

The Integrin-Mediated ILK-Parvin-aPix Signalling Axis Controls Differentiation in Mammary Epithelial Cells.

Rooney, N., Wang, P., Brennan, K., Gilmore, A., & Streuli, C.

Journal of Cellular Physiology. 2016. Publication link: 7368e317-26fe-44fd-8f79-222d4e8c06fa

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

Maguire, S. L., Peck, B., Wai, P. T., Campbell, J., Barker, H., Gulati, A., Daley, F., Vyse, S., Huang, P., Lord, C. J., Farnie, G., Brennan, K., Natrajan, R.

Journal of Pathology. 2016. 240(3), 315-328.

Increased peri-ductal collagen micro-organization may contribute to raised mammographic density.

Mcconnell, J., O'Connell, O. V., Brennan, K., Weiping, L., Howe, M., Joseph, L.,  Knight, D., O'Cualain, R., Lim, Y., Leek, A. , Waddington, R., Rogan, J., Astley, S. M., Gandhi, A., Kirwan, C. C., Sherratt, M.J., Streuli, C.

Breast Cancer Research. 2016. 18, [5].

Oncogenic activation of FAK drives apoptosis suppression in a 3D-culture model of breast cancer initiation.

Walker, S., Foster, F., Wood, A., Owens, T., Brennan, K., Streuli, C., & Gilmore, A.

Oncotarget. 2016. [Epub ahead of print]

Anti-estrogen Resistance in Human Breast Tumors Is Driven by JAG1-NOTCH4-Dependent Cancer Stem Cell Activity.

Simões BM, O'Brien CS, Eyre R, Silva A, Yu L, Sarmiento-Castro A, Alférez DG, Spence K, Santiago-Gómez A, Chemi F, Acar A, Gandhi A, Howell A, Brennan K, Rydén L, Catalano S, Andó S, Gee J, Ucar A, Sims AH, Marangoni E, Farnie G, Landberg G, Howell SJ, Clarke RB.

Cell Rep. 2015 Sep 29;12(12):1968-77. doi: 10.1016/j.celrep.2015.08.050.

Wnt-Notch signalling crosstalk in development and disease.

Collu, G., Hidalgo-Sastre, A. & Brennan, K (2014).

Cell Mol Life Sci, 71(18), 3553-67. eScholarID:246346 | PMID:24942883 | DOI:10.1007/s00018-014-1644-x

Phosphorylation of the proapoptotic BH3-only protein bid primes mitochondria for apoptosis during mitotic arrest.

Wang, P., Lindsay, J., Owens, T., Mularczyk, E., Warwood, S., Foster, F., Streuli, C., Brennan, K. & Gilmore, A (2014).

Cell Rep, 7(3), 661-71. eScholarID:246347 | PMID:24767991 | DOI:10.1016/j.celrep.2014.03.050

Aurora a kinase regulates mammary epithelial cell fate by determining mitotic spindle orientation in a notch-dependent manner.

Regan, J., Sourisseau, T., Soady, K., Kendrick, H., McCarthy, A., Tang, C., Brennan, K., Linardopoulos, S., White, D. & Smalley, M (2013).

Cell Rep, 4(1), 110-123. eScholarID:201736 | PMID:23810554 | DOI:10.1016/j.celrep.2013.05.044

Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming.

Schellenberg, B., Wang, P., Keeble, J., Rodriguez-Enriquez, R., Walker, S., Owens, T., Foster, F., Tanianis-Hughes, J., Brennan, K., Streuli, C. & Gilmore, A (2013).

Mol Cell, 49(5), 959-71. eScholarID:198503 | PMID:23375500 | DOI:10.1016/j.molcel.2012.12.022

Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-a status.

Harrison, H., Rogerson, L., Gregson, H., Brennan, K., Clarke, R. & Landberg, G (2013).

Cancer Res, 73(4), 1420-33. eScholarID:198501 | PMID:23248117 | DOI:10.1158/0008-5472.CAN-12-2505

Combining Notch inhibition with current therapies for breast cancer treatment.

Brennan, K. & Clarke, R (2013).

Ther Adv Med Oncol, 5(1), 17-24. eScholarID:198500 | PMID:23323144 | DOI:10.1177/1758834012457437

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer.

Eccles, et al (2013).

Breast Cancer Res, 15(5), R92. eScholarID:246345 | PMID:24286369 | DOI:10.1186/bcr3493

Dishevelled limits Notch signalling through inhibition of CSL.

Collu, G., Hidalgo-Sastre, A., Acar, A., Bayston, L., Gildea, C., Leverentz, M., Mills, C., Owens, T., Meurette, O., Dorey, K. & Brennan, K (2012).

Development, 139(23), 4405-4415. eScholarID:198502 | PMID:23132247 | DOI:10.1242/dev.081885

Specific ß-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells.

Jeanes, A., Wang, P., Moreno-Layseca, P., Paul, N., Cheung, J., Tsang, R., Akhtar, N., Foster, F., Brennan, K. & Streuli, C (2012).

The Journal of biological chemistry, 287(29), 24103. eScholarID:177356 | PMID:22511753 | DOI:10.1074/jbc.M112.360834

Density of human bone marrow stromal cells regulates commitment to vascular lineages.

Whyte, J. L., Ball, S. G., Shuttleworth, C. A., Brennan, K., & Kielty, C. M. (2011).

Stem Cell Research. doi:10.1016/j.scr.2011.02.001.

Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor.

Harrison, H., Farnie, G., Howell, S. J., Rock, R. E., Stylianou, S., Brennan, K. R., Bundred, N. J., and Clarke, R. B. (2010)

Cancer Res 70, 709-18

Keith Brennan's research centres on the role developmental signalling pathways play in the initiation of cancer. The majority of human cancers arise in epithelial tissues such as the colon, skin and breast. These organs turnover constantly, with damaged cells being removed by apoptosis and replaced by the controlled division and differentiation of stem cells. This process is regulated by a network of signalling pathways that are also responsible for the differentiation of the organs during embryogenesis. During the initial stages, most tumours closely resemble the tissue that they arise from suggesting that disruption of this developmental signalling network is the initial driving force in tumour formation. The aberrant activation of Wnt signalling in nearly all colorectal cancers provides a clear example of this, as the pathway also plays a pivotal role in regulating stem cell self renewal and differentiation during normal colonic development and turnover. Consequently a thorough understanding of the development of a tissue or organ will provide important insights into the changes in cell behaviour that lead to tumour initiation. Furthermore this is likely to lead to the development of rational and novel therapies to treat early stage cancers when the likelihood of cure is significantly greater.

In particular his group has focused on the role the Notch signalling pathway plays in human breast cancer. This pathway is essential for multiple aspects of metazoan development, and they have shown that elevated signalling through the pathway plays a significant role in the aetiology of human breast cancer. They observe an accumulation of NICD, the active form of Notch, in a wide variety of invasive breast cancer cell lines and tissue samples. Increased Notch signalling is also seen in ductal carcinoma in situ (DCIS) lesions, a pre-invasive form of breast cancer, where it predicts a significantly increased risk of recurrence (work done in collaboration with Gillian Farnie, Rob Clarke and Nigel Bundred). This also suggests that activation of the pathway is an early change in tumour progression. Furthermore his group has shown that this increase in Notch signalling plays a causal role in tumour formation, as blocking the pathway reverts the tumourigenic phenotype of breast cancer cell lines.

To understand how this increase in Notch signalling leads to cellular transformation and, hence, tumour formation, his group has examined its effects on mammary epithelial cell behaviour. Although they do observe a reduction in cell adhesion leading to a change in morphology and decreased cell proliferation, the most significant change is a profound apoptotic resistance. This includes a greater resistance to several DNA damaging chemotherapeutic drugs. Mechanistically, this apoptotic resistance occurs through the secretion of a paracrine factor that activates Akt. The increase in Akt signalling subsequently prevents the activation of p53 in response to DNA damage through the ASK1/JNK pathway.

Current experiments in the laboratory seek to identify the Akt-activating secreted factor induced by Notch signalling. They are also looking to identify reliable markers of Notch signalling in breast cancers and to determine how Notch signalling is activated. In the longer term, they wish to determine the therapeutic potential of targeting Notch signalling in the treatment of breast cancer.

Current & Future Projects

• Determining the molecular mechanism by which Dishevelled inhibits Notch signalling and its relevance to cell fate decisions in vertebrates (Anna Collu and Silvia Oliveira)
• Elucidating the points crosstalk between the Notch and Wnt pathways that limit Wnt signalling (Ana Hidalgo Sastre)
• Identifying the secreted factor induced by Notch signalling that activates Akt in human breast cancer
• Understanding how elevated EDAR signalling promotes the transformation of breast epithelial cells

Recent discoveries

Notch signalling prevents chemotherapy-induced apoptosis in breast cancer by activating Akt

The Notch signalling pathways is aberrantly activated in most human breast cancers. Our previous work has shown that this increase in Notch signalling induces a profound resistance to many apoptotic stimuli including chemotherapeutic agents. We have now shown that the apoptotic resistance is due to the activation of Akt by a Notch-induced secreted factor. Akt signalling then prevents the activation of p53 following treatment with DNA-damaging chemotherapeutic agents. Mechanistically this due to the phosphorylation of ASK1 by Akt blocking ASK1/JNK signalling and therefore the JNK mediate phosphorylation and activation of p53.

Elevated EDAR signalling in the mammary gland leads to focal tumours that display extensive squamous metaplasia

EDAR signalling is required for the normal development of several skin appendages, such as hair and teeth. We have recently shown that EDAR signalling plays a significant role in the development of many skin-derived glands including the mammary gland. We have also found that elevated EDAR signalling leads to supernumerary nipples in female mice, ductal development in males, and in focal mammary tumours in breeding female mice. The tumours that develop all display extensive squamous metaplasia. Interestingly, similar phenotypes are seen when Wnt signalling is elevated and EDAR has been shown to regulate Wnt gene expression during hair follicle development.

Dishevelled attenuates Notch signalling by promoting the degradation of RBP-J?

Work in Drosophila has suggested that Dishevelled, a component of the Wnt signalling pathway, can inhibit Notch signalling. We have shown that a similar crosstalk also occurs in vertebrates. Mechanistically the crosstalk is mediated by an interaction between Dishevelled and RBP-J?/CBF1, the transcription factor at the base of the Notch pathway, which promotes the proteosomal degradation of RBP-J?/CBF1.

Meurette, O., Stylianou, S., Rock, R., Collu, G. M., Gilmore, A. P., and Brennan, K. (2009) Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells, Cancer Res 69, 5015-22 PubMed link.

Collu, G. M., Meurette, O., and Brennan, K. (2009) Is there more to Wnt signalling in breast cancer than stabilisation of beta-catenin?, Breast Cancer Res 11, 105 PubMed link.

Chang, S. H., Jobling, S., Brennan, K., and Headon, D. J. (2009) Enhanced Edar signalling has pleiotropic effects on craniofacial and cutaneous glands, PLoS One 4, e7591 PubMed link.

Thompson, A., Brennan, K., Cox, A., Gee, J., Harcourt, D., Harris, A., Harvie, M., Holen, I., Howell, A., Nicholson, R., Steel, M., and Streuli, C. (2008) Evaluation of the current knowledge limitations in breast cancer research: a gap analysis, Breast Cancer Res 10, R26 PubMed link.

Oldershaw, R. A., Tew, S. R., Russell, A. M., Meade, K., Hawkins, R., McKay, T. R., Brennan, K. R., and Hardingham, T. E. (2008) Notch signaling through Jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells but must be switched off to complete chondrogenesis, Stem cells (Dayton, Ohio) 26, 666-74 PubMed link.

Kirton, J. P., Crofts, N. J., George, S. J., Brennan, K., and Canfield, A. E. (2007) Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease?, Circulation research 101, 581-9 PubMed link.

Farnie, G., Clarke, R. B., Spence, K., Pinnock, N., Brennan, K., Anderson, N. G., and Bundred, N. J. (2007) Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways, Journal of the National Cancer Institute99, 616-27 PubMed link.

Collu, G. M., and Brennan, K. (2007) Cooperation between Wnt and Notch signalling in human breast cancer, Breast Cancer Res 9, 105 PubMed link.

Stylianou, S., Clarke, R. B., and Brennan, K. (2006) Aberrant activation of notch signaling in human breast cancer, Cancer research 66, 1517-25PubMed link.

Langdon, T., Hayward, P., Brennan, K., Wirtz-Peitz, F., Sanders, P., Zecchini, V., Friday, A., Balayo, T., and Martinez Arias, A. (2006) Notch receptor encodes two structurally separable functions in Drosophila: a genetic analysis, Dev Dyn 235, 998-1013 PubMed link.

Hayward, P., Brennan, K., Sanders, P., Balayo, T., DasGupta, R., Perrimon, N., and Martinez Arias, A. (2005) Notch modulates Wnt signalling by associating with Armadillo/beta-catenin and regulating its transcriptional activity, Development (Cambridge, England) 132, 1819-30PubMed link.

Hardman, M. J., Liu, K., Avilion, A. A., Merritt, A., Brennan, K., Garrod, D. R., and Byrne, C. (2005) Desmosomal cadherin misexpression alters beta-catenin stability and epidermal differentiation, Molecular and cellular biology 25, 969-78 PubMed link.

Gonzalez-Sancho, J. M., Brennan, K. R., Castelo-Soccio, L. A., and Brown, A. M. (2004) Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin, Molecular and cellular biology 24, 4757-68 PubMed link.

Brennan, K., Gonzalez-Sancho, J. M., Castelo-Soccio, L. A., Howe, L. R., and Brown, A. M. (2004) Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins, Oncogene 23, 4873-84 PubMed link.

Brennan, K. R., and Brown, A. M. (2004) Wnt proteins in mammary development and cancer, Journal of mammary gland biology and neoplasia 9, 119-31 PubMed link.

Brennan, K., and Brown, A. M. (2003) Is there a role for Notch signalling in human breast cancer?, Breast Cancer Res 5, 69-75 PubMed link.

Martinez Arias, A., Zecchini, V., and Brennan, K. (2002) CSL-independent Notch signalling: a checkpoint in cell fate decisions during development?, Current opinion in genetics & development 12, 524-33 PubMed link.

Brennan, K., and Gardner, P. (2002) Notching up another pathway, Bioessays 24, 405-10 PubMed link.

Lawrence, N., Langdon, T., Brennan, K., and Arias, A. M. (2001) Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila, Curr Biol 11, 375-85 PubMed link.

Lawrence, N., Klein, T., Brennan, K., and Martinez Arias, A. (2000) Structural requirements for notch signalling with delta and serrate during the development and patterning of the wing disc of Drosophila, Development (Cambridge, England) 127, 3185-95 PubMed link.

Zecchini, V., Brennan, K., and Martinez-Arias, A. (1999) An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila, Curr Biol 9, 460-9 PubMed link.

Brennan, K., Klein, T., Wilder, E., and Arias, A. M. (1999) Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila, Developmental biology 216, 210-29 PubMed link.

Brennan, K., Baylies, M., and Arias, A. M. (1999) Repression by Notch is required before Wingless signalling during muscle progenitor cell development in Drosophila, Curr Biol 9, 707-10 PubMed link.

Brennan, K., Tateson, R., Lieber, T., Couso, J. P., Zecchini, V., and Arias, A. M. (1999) The abruptex mutations of notch disrupt the establishment of proneural clusters in Drosophila, Developmental biology 216, 230-42 PubMed link.

Arias, A. M., Brown, A. M., and Brennan, K. (1999) Wnt signalling: pathway or network?, Current opinion in genetics & development 9, 447-54 PubMed link.

Jacobsen, T. L., Brennan, K., Arias, A. M., and Muskavitch, M. A. (1998) Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila, Development (Cambridge, England) 125, 4531-40 PubMed link.

Klein, T., Brennan, K., and Arias, A. M. (1997) An intrinsic dominant negative activity of serrate that is modulated during wing development in Drosophila, Developmental biology 189, 123-34 PubMed link.

Brennan, K., Tateson, R., Lewis, K., and Arias, A. M. (1997) A functional analysis of Notch mutations in Drosophila, Genetics 147, 177-88PubMed link.