Rhost S, Hughes É, Ståhlberg A, Landberg G. Commentary: Sortilin Inhibition Limits Secretion-induced Progranulin-dependent Breast Progression and Cancer Stem Cell Expansion. J Human Clin Gen. (2019);1(1):6-9 Journal of Human & Clinical Genetics Commentary Open Access

Commentary: Sortilin Inhibition Limits Secretion-induced Progranulin- dependent Progression and Cancer Stem Cell Expansion Sara Rhost1, Éamon Hughes1, Anders Ståhlberg1,2,3, Göran Landberg1,2* 1Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden 2Department of Clinical Pathology and Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden 3Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden

The genetically conserved glycoprotein progranulin is a Article Info

Article Notes neurodegeneration, lysosomal function and tumorigenesis among Received: March 4, 2019 pleiotropic factor that is1,2 implicated in wound healing, , Accepted: March 26, 2019 other biological events . Progranulin is ubiquitously expressed and the fact that progranulin is genetically conserved and has a *Correspondence: wide-ranging expression pattern indicates important biological Dr. Göran Landberg, Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Cancer Center, functions. Progranulin acts as a holoprotein that can be cleaved University of Gothenburg, Gothenburg, Sweden; Email: into smaller peptide domains by the activity of , [email protected]. such as and cathepsin L3. Cleavage of progranulin occurs ©2019 Landberg G. This article is distributed under the terms of in the or within the . Interestingly, the Creative Commons Attribution 4.0 International License. cleaved granulin peptides are described to display different biological functions compared to the precursor progranulin in 4,5. In breast cancer, high progranulin expression is associated with reduced wounddisease-free healing, and inflammationoverall survival and6,7 neurodegeneration 8. Rhost and Hughes et al. and is identified as a prognostic marker for ERα positive breast cancer 9 cellsrecently showed reported high that constitutive progranulin secretion is differentially of progranulin secreted whereas by ERα positive and ERα negative breast cancer cell lines . ERα negative microenvironmental stress, such as low oxygen. These results imply thatthe progranulinsubtypes of breast secretion cancer fluctuated regulate in their ERα progranulin positive cells secretion during differently. Additional data obtained from Rhost et al. positive ductal carcinoma in situ (DCIS) samples with a hypoxic central area as an in vivo relevant model for hypoxia, revealed using ERα a clear gradient of progranulin expression towards the necrotic core (Figure 1). The increase of progranulin was further paralleled by

of the DCIS lesions validating the association between progranulin an increase of HIF1α and a decrease of ERα towards the inner etedge al.

cellsand hypoxia induced as estrogenwell as to independent ERα. These dataproliferation are in line via with activation Lu ofshowing cyclin thatD1 as overexpression well as tamoxifen of progranulin resistance 10in. CollectiveERα positive data MCF7 also support that hypoxia is not a solitary and local phenomenon but

progression and cancer treatment resistance. Growing evidence that thecan oxygenactually content influence of tumordistant tissue biological is an processes important including determinant tumor of metastasis11,12,13 reported by Rhost and Hughes et al. further highlight the importance of the findings et al., is that recombinant progranulin and the granulin domain A peptide inducedAn interesting breast cancer finding stem reported cell propagation, by Rhost andboth Hughes in vitro and in

Page 6 of 9 Rhost S, Hughes É, Ståhlberg A, Landberg G. Commentary: Sortilin Inhibition Limits Secretion-induced Progranulin-dependent Breast Cancer Progression and Cancer Stem Journal of Human and Clinical Genetics Cell Expansion. J Human Clin Gen (2019);1(1):7-9 vivo, induce m cells. These results demonstrate that progranulin and demonstrate that both progranulin and granulin peptides granulinusing A indeed ERα positive induce anda more ERα malignant negative form breast of cancerbreast most likelyore are metastaticinvolved in tumor cells. progression The general and findings future studies need to clarify the exact role for the granulins in the metastasis. The fact that granulin A mediates breast cancer development of metastatic disease. stemcancer cell with spreading a significantly suggests increased that enzymatic capacity cleavage to form To improve our understanding of how progranulin and of progranulin might play a role in rendering a more granulin induced breast cancer stem cell propagation, Rhost aggressive tumorigenesis. Although, previous results et al. further investigated the activation of several different published by Sayers et al. describe contrasting results with during progranulin and granulin treatment using an association of high secretion of Secretory Leukocyte a human phospho- array. Data indicated that both Inhibitor (SLPI) in highly metastatic cells14. SLPI progranulin and granulin A induced phosphorylation of nevertheless protects progranulin from enzymatic cleavage which indicates that inhibiting cleavage of progranulin potentseveral kinases,activator such compared as GSK3α/β to EGFR,granulin MSK1/2, A (Figure AMPKA1, 2). TheseAKT1/2/3, results and indicate mTOR, that where both progranulinprogranulin and was granulin a more

A substantially influence several major intracellular breastsignaling cancer pathways.15 where Interestingly, it was shown the that PI3K/AKT/mTOR AKT activated thepathway estrogen is implicated receptor pathwayin endocrine independently resistant ERα of estrogen positive

endocrine therapy was shown to overcome endocrine availability. Further, mTOR inhibitors in combination with

betweentherapy resistance. endocrine Thetherapy fact thatresistance progranulin and progranulin induces AKT/ via mTOR phosphorylation suggests a possible additional link

the DetailedAKT/mTOR analyses pathway. at single-cell level further revealed that progranulin and its receptor sortilin were both associated with signature for differentiation and Figure 1: Immunohistochemical stainings of ductal carcinoma in situ (DCIS) samples. Immunohistochemical analyses of DCIS demonstrating that differentiated cancer cells respond to samples were performed using HIF1α, Progranulin, CA-9, ERα progranulinepithelial-mesenchymal in a autocrine transitionsignalling pathway, (EMT) whereby properties the and Haematoxylin stainings. sortilin (SORT1) positive subpopulation gradually shifted

Figure 2: Progranulin and granulin A induce phospho-kinase expression in ERα positive MCF7 cells. MCF7 cells were treated with either vehicle (PBS) progranulin (PGRN, 1µg/ml) or granulin A (GRN A, 1µg/ml) for 50 minutes. Cell lysates (n:2) were then analysed using a phospho-kinase array system (R&D Systems). Data is expressed as mean pixel density.

Page 7 of 9 Rhost S, Hughes É, Ståhlberg A, Landberg G. Commentary: Sortilin Inhibition Limits Secretion-induced Progranulin-dependent Breast Cancer Progression and Cancer Stem Journal of Human and Clinical Genetics Cell Expansion. J Human Clin Gen (2019);1(1):8-9 from highly differentiated cell types to more cancer stem interest for future cancer therapy design with the potential cells-like cells9. Sortilin itself is reported to be associated to develop a unique cancer treatment based on selective with breast cancer aggressiveness16. Importantly, the targeting cancer stem cell propagation through sortilin. spreading of breast cancer stem cells induced by granulin A Sortilin is apart from being a receptor to progranulin also could be blocked by the orally bioavailable small molecule AF3846917, binding to sortilin. Further, elevated levels of IL619. In breast cancer, IL6 is associated with progranulin or granulin A impacted breast cancer stem cells described to bind with high-affinity to the proinflammatory similarly through the receptor sortilin, despite subtype of and treatment resistance20,21,22 further signifying the role of breast cancer. Progranulin and granulin A induced breast increasedsortilin in tumorbreast stage,cancer lymph progression. node infiltration, recurrence, cancer stem cell spreading resulted in a more aggressive Material and Methods

Single-cell qPCR analysis additionalcancer with single-cell a significant studies increase using in primary metastasis breast formation. cancer cellsTo further obtained validate from the surgery findings to clinically,determine we the performed relation Single cell analyses were performed as earlier described 22. The analyses of primary tumor samples were analysed 18 by differentiation and cancer stem cell markers as earlier according to the protocol described in Akrap et al. . establishedbetween progranulin,18 sortilin and subpopulations defined Human Phospho-Kinase Antibody Array positive breast cancer and corresponding axillary lymph node metastasis. In were total, analysed 184 individual using principal cells from component an ERα The phospho-kinase antibody array was performed analysis. SORT1 and GRN clustered with the differentiation according to the protocol provided by the manufacturer associated genes in the primary tumor (Figure 3A) and upon metastatic spread, the expression of SORT1, GRN and a number of proliferative markers nevertheless clustered minutes(R&D systems). were analysedCell lysates for from phosphorylation MCF7 cells treated of several with with cancer stem cell associated genes (Figure 3B). These differentprogranulin kinases (1 µg/ml) using or the granulin human A (1 phospho-kinase µg/ml) for 50 results suggest that the SORT1 expressing population in the antibody array. primary tumor was associated with highly differentiated cells, whereas SORT1 expressing cells during disease Immunohistochemistry progression associated with genes related to cancer stem cell features. Collective data concerning progranulin and sortilin thickTissue sections. sections Immunohistochemistry were fixed with 4 % phosphate was performed buffered presence in cancer cells and the now reported link to formaldehyde, embedded in paraffin and cut into 4.5 µm cancer stem cells and cancer progression could be of using DAKO Autostainer LINK 48 using Envision FLEX+ detection system. Briefly, deparaffinized sections were

Figure 3: Differentiated sortilin positive cells change their transcriptional signature during disease progression in patient derived tumor samples. Single cell analyses of GRN and SORT1 (black) and sets of differentiation (blue), proliferation (green) and cancer stem cell -related (red) transcripts and the parallel cluster principal component analysis of (a) a primary ERα positive breast cancer and (b) the corresponding axillary lymph node metastases.

Page 8 of 9 Rhost S, Hughes É, Ståhlberg A, Landberg G. Commentary: Sortilin Inhibition Limits Secretion-induced Progranulin-dependent Breast Cancer Progression and Cancer Stem Journal of Human and Clinical Genetics Cell Expansion. J Human Clin Gen (2019);1(1):9-9

6. Survival Factor GP88 (Progranulin) in the of Breast Cancer and DIVA antigen retrieval pH 6.2, followed by blocking Tkaczuk KR, Yue B, Zhan M, et al. Increased Circulating Level of the subjected to antigen retrieval by high pressure cocking Patients When Compared to Healthy Subjects. Breast Cancer (Auckl). with 3% hydrogen peroxide and incubation with primary 7. 2011; 5: 155-62. expression is associated with increased risk of recurrence in breast antibody against HIF1α (ab82832, abcam), progranulin cancerSerrero patients G, Hawkins diagnosed DM, Yue with B, et estrogenal. Progranulin receptor (GP88) positive tumor invasive tissue (MA1-187, Thermo scientific), CA-9 (ab15086, Abcam), ER α (M7047, Dako) at RT for 1 hour. For signal amplification 8. ductalKoo DH, carcinoma. Park CY, Lee Breast ES, etCancer al. Progranulin Res. 2012; as 14(1): a prognostic R26. biomarker EnVision™ FLEX+ rabbit linker (SM805, DAKO) and for breast cancer recurrence in patients who had receptor- EnVision™ FLEX+ mouse linker (SM804, DAKO) was used, respectively. Further EnVision FLEX/HRP visualization 9. positiveRhost S, tumors: Hughes a cohortÉ, Harrison study. PLoSH, et One. al. 2012;Sortilin 7(6): inhibition e39880. limits reagent EnVision™ FLEX/HRP secondary antibody-coated secretion-induced progranulin-dependent breast cancer progression polymer peroxidase complexes (#SM802, DAKO), followed by DAB substrate/chromogen (DAKO) was used. Slides 10. and cancer stem cell expansion. Breast Cancer Res. 2018; 20(1): 137. were counterstained with hematoxylin (DAKO). Stained Lu R, Serrero G. Mediation of estrogen mitogenic effect in human sections were scanned by Leica SCN400 scanner at 20X breast cancer MCF-7 cells by PC-cell-derived (PCDGF/ and evaluated by the automated image analysis Definiens 11. granulin precursor). Proc Natl Acad Sci U S A. 2001; 98(1): 142-7. DeveloperAcknowledgments XD tissue studio program. Bellot G, Garcia-Medina R, Gounon P, et al. Hypoxia-induced We thank the patients from Sahlgrenska University is mediated through hypoxia-inducible factor induction of BNIP3 and 12. BNIP3L via their BH3 domains. Mol Cell Biol. 2009; 29(10): 2570-81. Hospital who donated samples for this research and the Controversial data concerning time frames and biological pathology and surgery departments for patient consent Bayer C, Vaupel P. Acute versus chronic hypoxia in tumors: and sample collection. We thank our colleagues Pernilla 13. consequences.Rofstad EK, Gaustad Strahlenther JV, Egeland Onkol. TA, 2012; et al. 188(7): Tumors 616-27. exposed to acute Gregersson, Karoline Berger, Daniel Andersson and Ylva cyclic hypoxic stress show enhanced angiogenesis, perfusion and

14. metastaticSayers KT, Brooksdissemination. AD, Sayers Int JTJ, Cancer. et al. 2010;Increased 127(7): secretory 1535-46. leukocyte FundingMagnusson for their technical research contribution. protease inhibitor (SLPI) production by highly metastatic mouse This work was supported by grants from Knut and Alice Wallenberg Foundation; Wallenberg Centre for 15. breast cancer cells. PLoS One. 2014; 9(8): e104223. Paplomata E, O’Regan R. New and emerging treatments for estrogen Gothenburg, Sweden; Swedish Cancer Society (2016-486 receptor-positive breast cancer: focus on everolimus. Ther Clin Risk Molecular and Translational Medicine, University of 16. Manag.Roselli 2013;S, Pundavela 9: 27-36. J, Demont Y, et al. Sortilin is associated with and 2016-438); Swedish Research Council (521-2012- breast cancer aggressiveness and contributes to tumor cell adhesion 5716, 16-06074 and 2017-01392); the Swedish state under the agreement between the Swedish government 17. and invasion. Oncotarget. 2015; 6(12): 10473-86. and the county councils; the ALF-agreement (7211091 Schrøder TJ, Christensen S, Lindberg S, et al. The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family 18. sorting receptor Sortilin. Bioorg Med Chem Lett. 2014; 24(1): 177-80. atand University 716321); of VINNOVA; Gothenburg; Assar Johan Gabrielssons Jansson Foundation Research Cancer Stem Cell Populations Based on Single-Cell Analyses of Foundation; BioCARE National Strategic Research Program FunctionallyAkrap N, Andersson Enriched D, Stem Bom andE, et Progenitor al. Identification Pools. Stem of Distinct Cell Reports. Breast for Cancer Research; Wilhelm and Martina Lundgren 19. 2016; 6(1): 121-36. ReferencesFoundation for Scientific Research. Mortensen MB, Kjolby M, Gunnersen S, et al. Targeting sortilin in 1. immune cells reduces proinflammatory cytokines and . 20. J Clin Invest. 2014; 124(12): 5317-22. De Muynck L, Van Damme P. Cellular effects of progranulin in health 2. and disease. J Mol Neurosci. 2011; 45(3): 549-60. Knupfer H, Preiss R. Significance of interleukin-6 (IL-6) in breast 21. cancerSalgado (review). R, Junius Breast S, Benoy Cancer I, et Res al. CirculatingTreat. 2007; interleukin-6 102(2): 129-35. predicts Kao AW, McKay A, Singh PP, et al. Progranulin, lysosomal regulation and survival in patients with metastatic breast cancer. Int J Cancer. 2003; 3. neurodegenerative disease. Nat Rev Neurosci. 2017; 18(6): 325-333. Lee CW, Stankowski JN, Chew J, et al. The lysosomal protein cathepsin 22. 103(5):Zhang GJ, 642-6. Adachi I. Serum interleukin-6 levels correlate to tumor 4. L is a progranulin protease. Mol Neurodegener. 2017; 12(1): 55. characterization of two cysteine-rich growth-modulating . progression and prognosis in metastatic breast carcinoma. Anticancer Shoyab M, McDonald VL, Byles C, et al. Epithelins 1 and 2: isolation and 23. 5. Proc Natl Acad Sci U S A. 1990; 87(20): 7912-6. Res. 1999; 19(2B): 1427-32. Ståhlberg A, Rusnakova V, Kubista M. The added value of single-cell epithelinTolkatchev modules D, Malik with S, unique Vinogradova functional A, activities. Wang P, Protein et al. Sci. Structure 2008; dissection of human progranulin identifies well-folded granulin/ profiling. Briefings in Functional Genomics. 2013; 12(2): 81-89. 17(4): 711-24.

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