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A “Seed Pre-Selection” Hypothesis of Breast Cancer Metastasis Xiang (Shawn) Zhang Lester and Sue Smith Breast Center Baylor College of Medicine
Metastasis: a multi-step biological processPrimary tumor growth Invasion and intravasation Survive in circulation Extravasation ColonizationPrimary tumor Metastasis A sequence of Darwinian selection processes?
Metastasis organ tropism and latencyMetastasis behavior can be encoded in primary tumors – A conundrum:How and why do pro-metastasis traits arise in primary tumors?
We set out to understand the basis of metastasis organ- tropism and latency using integrative approaches. Bone metastasisClinical samples Years to decades Lung metastasis Brain metastasis Primary tumor transcriptional profile Pathways linked to clinical outcome: 1. Is there metastasis? KRAS SRC TGFß 2. To where? 3. When? WNT MYC E2F STAT3 NFkB … Signaling pathway signatures obtained from cell line models Experimental validation: are these pathways functionally driving metastasis?
Association of SRS with overall and late-onset bone relapse Bone relapse by SRS status 15 * 1.0 SRS Hazard ratio ER 0.8 ** * SRS+ breast tumors: 10Probability 0.6 ~90% of ER+ tumors ~50% of HER2+ tumors 0.4 ~25% of TN tumors 5 ** *** *** 0.2 SRS+ SRS- 0.0 0 >0 >12 >24 >36 >48 >60 0 40 80 120 160 bone metastasis onset Bone metastasis-free survival (months) The association is unique between SRS and bone metastasis, and is independent of other conventional clinical parameters (treatment, stage, genomic grades …).
Src knock-down in aggressively metastatic cells inhibits bone metastasis (MDA-MB-231 xenograft model)Intracardiac Bone injection metastasis 35 days flux at hind limbs x10–5 10 p=0.002 Normalized photon Control 8 * * 6 4 Src RNAi 2 0 Src RNAi + Src RNAi Control Src Rescue Src-Rescue * MDA231-BoM1833 No effect of Src RNAi on mammary tumor growth or on lung metastasis. Knock-down of Fyn and Yes did not have similar effects.
Dasatinib, a drug that inhibits Src, suppresses bone colonization Day 35 after injection Day 35 after injection Normalized photon flux at Normalized photon flux at p=0.002 p=0.61 hind limb (x103) hind limbs (x103) 10 6 p=0.054 8 4 p=0.001 p=0.011 6 4 2 2 0 0 Day 14 Day 0 Day 7 Mock Dasatinib Src rescue Wild type Dasatinib c-Src resistant c-Src Dasatinib Start MDA231-BoM1833 MDA231-BoM1833 Src RNAi No effect of dasatinib on lung-metastasis.
Src knock-down in aggressively metastatic cells compromises their survival in the bone marrow Control Src RNAi Dasatinib Src Rescue p=0.02TUNEL 20 p=0.005 p=0.003 Percent TUNEL+ cells 15 10 5Ki67 0 Control Src RNAi Dasatinib Rescued Bone met microenvironment Cancer Cells Bone Src inhibition has no effect on degradation osteoclast mobilization. Osteoclasts
Cytokines enriched in the human bone metastasis microenvironment58 Human breast cancer metastasis samples Bone Lung Liver Brain BMP2: bone morphogenetic protein 2 IGF2: insulin-like growth factor 2 CLEC11A: C-type lectin domain family 11A; SCGF CXCL12: chemokine C-X-C motif 12; SDF1 CXCL14: chemokine C-X-C motif 14 GMFG: glia maturation factor, gamma IGF1: insulin-like growth factor 1 JAG1: jagged 1 NOV: nephroblastoma expressed; IGFBP9 PDGFA: platelet-derived growth factor alpha PGF: placental growth factor PXDN: peroxidasin homolog SPP1: secreted phosphoprotein 1; osteopontin TGFB1: transforming growth factor, beta 1 TGFB3: transforming growth factor, beta 3 TNFSF10: tumor necrosis factor family 10; TRAIL VEGFC: vascular endothelial growth factor C -2 0 2 4 The enrichment of many of these cytokines are recapitulated in xenografted metastases.
Src mediates CXCL12 and IGF1-induced survival and antagonizes TRAIL-induced apoptosis Control Src RNAi Rescue ns ** * ** ** Percentage Viable (Day 5) 60 ** ** 60 Percent Viable (Day 3) 50 50 40 40 30 30 20 20 10 10 0 0 No serum No serum + No serum + No Serum No Serum + CXCL12 IGF1 TRAIL
Model: Src confers survival advantages to cancer cells in the bone microenvironment. Bone marrow survival factors IGF1 CXCL12 TRAIL IGF1R CXCR4 DR4/5 Src . . . Survival . . . SRS+ breast cancer cell Zhang et al., 2009 Competence Competence to infiltrate Competence to colonize to survive Breast Bone carcinoma marrow Bone macrometastasis (years to decades) CXCR4, PTHrP, IL11, MMP1, OPN How and why is Src activated in primary tumors? Whether can we find a better indicator of the existence of latent bone metastasis?
SRS associates with ER and ERBB2 statuses Bone relapse SRS ERERBB2 Negative Positive 25 ER+ ER-/ERBB2+ Percentage of tumors (%) ER-/ERBB2- 20 15 10 5 0 SRS score (scaled between -1 and 1)
To search for Src activation mechanism in the ER- /ERBB2- subtypeApproach: compare SRS+ tumors with SRS- tumors and examine thegenes that associate with SRS (but not part of the SRS itself). EMC-MSK-615 TRANSBIG UPPSALA STOCKHOLM ER-/ERBB2- tumors 3 9 7 4 0 4 3 1 1 4 1 8 5 8 SRS- v.s. SRS+ CXCL12 CXCL14 IGF1 IGF2 15 10 -log10p 5 0
A same group of cytokines enriched in SRS+ primary tumors and bone metastases ER-/SRS+ ER-/SRS++4 58 Human breast cancer metastasis samples IGF1 Bone Lung Liver Brain+2 IGF2 CXCL14 CXCL12 CXCL120 IGF1 CXCL14 IGF2-2 overexpressed in bone ERBB2 Other cytokines metastases IGF1 IGF2 CXCL12 CXCL14 ERBB2 ER-/ERBB2- ER-/ERBB2+
ER-/ERBB2- subtype: CXCL12 and IGF1 activate AKT in a Src- dependent manner MDA231 CN34 MDA231 CN34 CXCL12 Control Rescued Src RNAi Control Src RNAi Rescued IGF1CXCL12 Dasatinib P-AKT pAKT AKT pSrc c-Src AKTCXCR4 Src Tubulin Tubulin 1. CXCL12 and IGF1 do NOT directly activate Src. 2. CXCL12 and IGF1 promote cell survival in a Src dependent manner. 3. Cancer cells do NOT express CXCL12 and IGF1.
ER-/ERBB2- Subtype: the hypothesis of “Metastasis seed pre-selection” 58 Human breast cancer metastasis samples ER-/SRS+ ER-/SRS+ Bone Lung Liver Brain Primary tumors Bone marrow Survival upon arrival v Colonization CXCL12 IGF1 CXCL12 A IGF1 CXCR4 IGF1R B Src C PI3K/AKT … Enrichment of Bone marrow survival factors Src activity IGF1 CXCL12 TRAIL IGF1R CXCR4 DR4/5 Src . . * * . Survival * * * . . . * * SRS+ breast cancer cell
Long term incubation with CXCL12/IGF1 causes a colony expansion process in vitro Low serum with CXCL12/IGF1 MDA-MB-231 cells Low serum only Parental cells Low serum Colony expansion Subpopulations + CXCL12, IGF1 Regular medium 3 ~ 9 weeks 1 ~ 2 weeks
Long term incubation with CXCL12/IGF1 expanded colonies with higher Src activity MDA-MB-231 (Passage: 3) Parental IGF1 (ng/ml): 0 3 10 30 100 PCXCL12 (ng/ml): 0 10 30 100 300 P IGF1(10ng/ml) CXCL12 (30ng/ml) 0.4 intensity Relative 0.2 0 pTyr416-Src: Total-Src: Tubulin: 0 101 102 103 104 105 pY416-Src Western Phospho-Flow
Long term incubation with CXCL12/IGF1 led to more boneNormalized photon flux at hind limbs metastasis 100 Parental p = 0.0003 p = 0.021 p < 0.0001 0/0 10 10/30 1 P 0 10 100 P 0 30 300 100/300 IGF1 (ng/ml) CXCL12 (ng/ml) Bioluminescence Hind limbs Vertebrae H&E H&E No increase in lung metastasis bone specific The effect on bone metastasis was also seen in another cell line: CN34
In vitro selected MDA231 derivatives have not acquired osteolytic competence Parental 0-0 10-30 10 - 30 BoM-1833 BoM-1833 10-30: MDA231 derivatives selected by in vitro cultivation with IGF1 and CXCL12 (10ng/ml and 30ng/ml, respectively).Bone metastasis genes identifiedthrough in vivo selection (Kang et BoM-1833: MDA231 derivatives extracted from established boneal., 2003) lesions (Kang et al., 2003)
Seed pre-selection in the indolent metastasis systems ER- pleural effusion samples CN37 CN34 0+0 10 + 30 0 + 0 10 + 30 P P pTyr416-Src Tubulin p = 0.014 25(Human B2M / Mouse Actb) 101 Relative mRNA expression 20 Mock Normalized photon Dasatinib flux at hind limbs 100 15 ×10-5 p = 0.0007 10 10-1 5 10-2 0 0-0 10-30 0 10 20 30 CN34 Days after injection
ER-/ERBB2- Subtype: the hypothesis of “Metastasis seed pre-selection” Primary tumors Bone marrow Survival upon ? ? arrival v Colonization CXCL12 IGF1 CXCL12 A IGF1 CXCR4 IGF1R B Src C PI3K/AKT … Enrichment of Src activity
Carcinoma-associated fibroblasts (CAFs) are enriched in SRS+ tumors * CAF over-expressing genes Breast cancer metastases 1.0 0.8 CAF SRS Probability 0.6 p = 0.025 signature 6 associated (77) genes (44) 0.4 CAF+ 0.2 CAF- 1 SPARCL IGFBP4 IGF1 FOS CXCL14 CXCL12 0 0 50 100 150 Bone metastasis free survival (Month)
MSCs differentiate into CAFs and provide CXCL12/IGF1 in mammary tumors MSCs MDA231 MDA231 aloneMDA231 and hMSC co-injection aSMA IGF1 CXCL12
The hypothesis of “Metastasis seed pre-selection” Primary tumors Bone marrow CAF Survival upon arrival v MSC Colonization CXCL12 IGF1 CXCL12A IGF1 CXCR4 IGF1RB SrcC PI3K/AKT… Enrichment of Src activity
Summary• The chance that a patient carries latent bone metastasis may be predictable based on primary tumor profiles (stromal content and Src activity).• We provide one possible answer to the metastasis progression puzzle: – When the microenvironment of a primary tumor resembles that of a distant organ, it will pre-select cancer cells predisposed to colonize that organ.
Acknowledgement• PI: Joan Massagué • Don Nguyen• Qiongqing Wang • Paula Bos• Xin Jin • David Padua • Johansson Baez• Larry Norton• Clifford Hudis • Karen Xi• William Gerald • Qing Chen• Neal Rosen • Weiping Shu• John Foekens• Marcel Smid • And other Massagué Lab members• NIH: K99CA151293