TGFβ/BMP/Smad signaling pathway

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1 TGFβ/BMP/Smad signaling pathway Dr. Jean Jacques Lebrun Professor of Medicine, McGill University Health Center, Cancer Research Program Associate Dean, Graduate & Postdoctoral Studies McGill University TGF Superfamily Members and Structure Monomer Dimer 1

2 TGFβ targets and its actions in mature tissues -Cell Growth -Apoptosis -Immortalization Tumor Suppression -Immunosuppression -Angiogenesis -Cell Adhesion -Cell migration -ECM/metalloproteinases -Invasion/metastasis Tumor Progression Nature Reviews Cancer 3, (2003) Mammary Gland Placenta hcg Progesterone GRF Gonads Cell growth Milk proteins Estrogens Androgens Bone marrow Erythropoiesis Cell growth Activins Pituitary FSH, LH, GnRHR ACTH, GH, PRL Cell growth Embryos Dorsal mesoderm Blood Tumor Cells Cell growth Apoptosis Cell growth Apoptosis Liver Cell growth Apoptosis 2

3 Selected Causes of Potential Years of Life Lost (PYLL), Canada Males Females Cancer Diseases of the heart Respiratory diseases Non-motor Vehicle accidents Cerebrovascular disease Suicide Motor vehicle accidents Diabetes Perinatal causes Congenital anomalies Cirrhosis HIV PYLL (in thousands) Myostatin null mice Sections of distal hindlimb stained with haematoxylin and eosin (Nature , 1997) 3

4 Multiple TFs Co-activators CBP/p300 Co-repressors (myc, Snail, Sno, Ski Evi1) proto-oncogenes - malignant transformation Lebrun, JJ. ISRN Molecular Biology, vol. 2012, Article ID , 28 pages, Lebrun, JJ. ISRN Molecular Biology, vol. 2012, Article ID , 28 pages,

5 Loss or mutation of TGF signaling in human cancers TGF as tumor suppressor Tumor cells secrete high level TGFβ Humbert L, Neel JC, Lebrun JJ, (2011). Trends in Cell & Molecular Biology. Neel JC, Humbert L, Lebrun JJ (2012). Médecine Sciences Amérique, Vol 1, No 3. Lebrun, JJ. ISRN Molecular Biology, vol (2012), Article ID , 28 pages, TGFβ and Tumor Suppression: 1- Cell Cycle Arrest 2- Apoptosis 3- Immortality Lebrun, JJ. ISRN Molecular Biology, vol. 2012, Article ID , 28 pages,

6 TGFβ and Tumor Suppression 1- Cell Cycle Arrest 2- Apoptosis 3- Immortality Lebrun, JJ. ISRN Molecular Biology, vol. 2012, Article ID , 28 pages, TGFβ-mediated transcription of its pro-apoptotic targets is dependent on E2F1 Korah J, et al., Cell Death and Disease, Nature Publishing Group, Vol 3. pp 407 6

7 TGFβ induces DNA binding of prb-pcaf-e2f1 complex to the apoptotic gene promoters Caspase Activity Luminescence (RLU) TGFβ treatment (hours) ctl sirna P/CAF sirna Korah J, et al., Cell Death and Disease, Nature Publishing Group, Vol 3. pp 407 TGFβ and Tumor Suppression: 1- Cell Cycle Arrest 2- Apoptosis 3- Immortality Acquired abilities of cancer cells Activin/TGF Cell Cycle - + Apoptosis - Immortalization Immunosuppression Tumor Suppression Cell, Vol. 100, 57 70,

8 Human Somatic Cells Limited proliferative potential (Hayflick limit) Telomeres shortening Loss of structural integrity Loss of protection vs exonucleases Cellular senescence and crisis Human Cancer Cells Infinite Replication and Immortalization Reactivation Telomerase activity Addition telomeric repeat (Chromosome stabilty, protection exonucleases) Cancer Diagnosis marker Reverse Transcriptase RNA template Protein htert Add telomeric repeat (TTAGGG) TGF Represses htert Gene Expression RNA Protein htert Gene promoter Lacerte et al., Cell Signal, 20(1):

9 Lebrun, JJ. ISRN Molecular Biology, vol. 2012, Article ID , 28 pages, TGFβ-mediated tumour suppression through E2F family members? E2F Family super mediators of TGFβ tumour suppressive effects Korah et al, Cell Death and Disease, 2012 Yang et al, Oncogene, 2008 Korah J, et al., J Biol Chem,

10 Breast Cancer Metastasis Stemness Drug resistance 10

11 Incidence Canadian statistics Year: 2014 Males Females Cases ,700 Incidence rank - 1 st Deaths 55 5,100 Death rank - 2 nd 5-year relative survival 79% 88% 62 Canadian women will be diagnosed with breast cancer every day (1/9) 14 Canadian women will die of breast cancer every day (1/27). A dozen signs of Breast Cancer 11

12 Staging and Grading for Breast Cancer Stage Stage Description Description (Tumor (Tumor size, size, Spreading spreading from from primary primary site) site) 0 There are two kinds of stage 0 breast cancer: Ductal carcinoma in situ (DCIS): Abnormal cells are in the lining of a milk duct and have not spread outside the duct. Lobular carcinoma in situ (LCIS): Abnormal cells are in the lining of a lobule. 1 Tumour is 2 cm or smaller and the cancer has not spread outside the breast. 2 Tumour is 2 to 5 cm, or cancer has spread to the lymph nodes, or both. 3 Cancer has spread to the lymph nodes and may have spread to nearby tissues such as the muscle or skin. 4 Cancer has spread to distant parts of the body. Grade description (Biopsy sample, growth vs normal cells) Grade Description (Biopsy sample, Growth compared to normal cells) Small aggressive tumors 15-20% metastatic tumor unknown origin 1 Low grade well differentiated - slow growing, less likely to spread 2 Moderate grade 3 High grade poorly or undifferentiated - tend to grow quickly, more likely to spread TNM (Tumor size, Lymph Node, Metastasis), T(1,2,3,4) 12

13 Stage TNM Description 5-year Survival 60 % 0 Tis N0 M0 Carcinoma in situ. No tumor is regional lymph nodes, No distant metastases I T1 N0 M0 Tumor is less than or equal to 2 centimeters, No tumor is regional lymph nodes, No distant metastases IIA T0 N1 M0 No evidence of primary tumor, metastases to movable ipsilateral T1 N1 M0 nodes, No distant metastases. T2 N0 M0 Tumor is less than or equal to 2 centimeters, metastases to movable ipsilateral nodes, No distant metastases. 99% 92% 82% IIB T2 N1 M0 T3 N0 M0 Tumor is between 2 and 5 centimeters, No tumor is regional lymph nodes, No distant metastases Tumor is between 2 and 5 centimeters, metastases to movable ipsilateral nodes, No distant metastases. 65% 33% IIIA T0 N2 M0 T1 N2 M0 T2 N2 M0 T3 N1, N2 M0 Tumor is over 5 centimeters, No tumor is regional lymph nodes, No distant metastases. No evidence of primary tumor, metastases to fixed ipsilateral nodes, no distant metastases. Tumor is less than or equal to 2 centimeters, metastases to fixed ipsilateral nodes, No distant metastases. 47% Tumor is between 2 and 5 centimeters, metastases to fixed ipsilateral nodes, no distant metastases. IIIB T4 Any N M0 Any T N3 M0 Tumor is over 5 centimeters, metastases to movable or fixed ipsilateral nodes, no distant metastases. Tumor extends to chest wall, any nodal involvement, no distant metastases. 44% Any primary tumor involvement, metastases to ipsilateral internal mammary nodes, no distant metastases. 5% IV Any T Any N M1 Any primary tumor involvement, any nodal involvement, distant metastases. 14%

14 Molecular Classification of Invasive Ductal Carcinoma Basal prognostic characters : larger tumor, higher grade, lymph node spread, and a higher rate of distant metastasis; highly invasive, resistant chemo and radiotherapies (enriched in cancer stem cells) Eroles et al., Cancer Treatment Reviews, Volume 38, Issue 6, October 2012, pp Vargo-Gogola and Rosen. (2007) Nat Rev Cancer 7: TGFβ Signaling Components are Highly Expressed in Basal-B like TNBC Cell Lines 14

15 p21 plays a central downstream of TGFβ-mediated cell migration and invasion TGFβ-induced Genes are Largely Involved in Cell Migration TGFß induces p21 expression in human metastatic breast cancer cells -- migration/invasion - metastasis? p21 expression correlates with poor survival in breast cancer patients Dai M, et al., Breast Cancer Res, 2012 Sep 20;14(5):R

16 p21 is required for TGFß-mediated cell migration Dai Me, Lebrun JJ., Breast Cancer Res, 2012 Sep 20;14(5):R127. p21 is required for TGFß-mediated cell invasion Matrigel (GFR) TGFβ FBS Dai M, Lebrun JJ, Breast Cancer Res, 2012 Sep 20;14(5):R

17 Silencing p21 prevents local tumor invasion Fat pad transplantation Control mice Breast cancer cells injected mice Wild-type invasive breast cancer cells P21-silcenced invasive breast cancer cells Dai Me, Lebrun JJ., Breast Cancer Res, 2012 Sep 20;14(5):R127. TGFβ Stemness Drug resistance 17

18 Cancer stem cells and Cancer Therapies Screen clipping taken: 1/12/2017 3:20 PM European Journal of Cancer (2012) 48, Identification and detection of CSCs in various cancers In vitro methods: Specific cell surface markers Tumorsphere formation assay Limiting dilution assay Expression of stemness markers In vivo method: Serial transplantation 18

19 Breast cancer stem cells (BCSCs) markers: CD44+/CD24 Xenograft model: Mammary fat pad injection of 20,000 CD44+CD24-cells and 20,000 CD44+CD24+Lineage+ cells in NOD/SCID mice. Six months later, the injection sites were examined by histology. BCSCs identified as a CD44 + /CD24 /low population display enhanced ability to initiate tumour growth. Xenograft model: Human breast cancers were transplanted orthotopically in the humanized cleared fatpad of NOD/scid mice ALDH+ cells generated a tumor ALDH- cells failed to generate tumors. overlap 1% or less of the total cancer cell population. These cells are highly enriched in tumorigenic capability, being able to generate tumors from as few as 20 cells. ALDH is a marker of malignant human mammary stem cells. Al-Hajj et al., PNAS Tumorsphere formation assay TGFβ-induced CD44high/CD24low BCSC populations in TNBC TGFβ-induced ALDH+ BCSC populations in TNBC Tian, Lebrun, Nature Scientific Reports, Jan

20 Cancer Stem Cell Regulation by TGFβ Cell cycle Regulators: TGFβ regulates cyclin D1 expression and CDK4 activity (prb) to promote metastasis Role of CDK4 in promoting stemness Blocking CDK4 or TGFβ will eliminate cancer stem cells (Palbociclib) Cyclooxygenase-2 (Cox2) enzymatic pathway: TGFβ regulates Cox2 expression and enzymatic activity (PGE2 production) Cox2 highly expressed in in TNBCs poor prognosis TGFβ/Cox2 regulate Cd44/24 and ALDH+ CSCs Blocking Cox2 eliminate cancer stem cells (Celecoxib) Cox2 is overexpressed in triple negative breast cancer Cox2 Inhibitor Celecoxib Targets Breast Cancer Stem Cells Tian, Lebrun, Nature Scientific Reports, Jan

21 CDK4 is overexpressed in triple negative breast cancer LY Dai, Lebrun, Nature Scientific Reports, Oct 2016 CDK4 Inhibitor Palbociclib Targets Breast Cancer Stem Cells LY Dai, Lebrun, Nature Scientific Reports, Oct

22 CDK4 Inhibitor Palbociclib Targets chemotherapy-resistant Breast Cancer Stem Cells Palbociclib: - approved FDA 2015 for ER+ breast tumors in combination with aromatase inhibitors (block estrogen) - next to be tested n TNBC patients and chemo-resistant clinical trials Dai, Lebrun, Nature Scientific Reports, Oct 2016 CDK4 Inhibitor Palbociclib blocks primary tumor growth Mouse model: NOD scid gamma (NSG) mice MDA-MB-231-luciferase cells - Flank injection 6 mice per group CDK4 Inhibitor Palbociclib blocks lung metastasis Normalized Luciferase intensity Control mice Palbociclib-treated mice Tian, Dai, Lebrun,

23 Thank You Questions? 23

Hormones and Cancer Research Unit Department of Medicine, McGill University Dr. Jean-Jacques Lebrun

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