Can we prevent metastasis?

Transcription

1 Can we prevent metastasis? A research example to translate from the bench to the bedside Diane Palmieri, Ph.D. Women s Cancers Section Laboratory of Molecular Pharmacology CCR, NCI

2 Some Basic Truths Most cancer patients succumb to direct or indirect effects of metastasis Direct organ compromise Consequences of treatment Paraneoplastic syndromes Most preclinical drug development is conducted in primary tumor models in mice The metastatic process has been considered undruggable and untestable. True? Is metastasis a valid therapeutic target?

3 What about a valid prevention target?

4 What is Metastasis? The spread of tumor cells resulting in the colonization of a distant site. Measured in humans by imaging, life span Measured experimentally by animal studies. There is no in vitro assay for metastasis. Measured in mice by injection of tumor cells, either orthotopically (spontaneous) or into the circulation (experimental)

5 Spontaneous Assay Spontaneous In the MFP Experimental Assay Experimental In the circulation - Tail Vein - Heart - Carotid Artery

6 An Example of a Preclinical Prevention Model for Metastases Day 0: Day 3: Injection of tumor cells Begin treatment with experiment therapeutic Day X: End experiment, remove tissues of interest for histological analysis

7 An Example of a Preclinical Treatment Model for Metastases Day 0: Day 3: Day 14: Day 28: Day x: Injection of tumor cells Mice randomized to 4 groups Group 1 - Vehicle treatment started Group 2 - Drug treatment started Group 3 - Drug treatment started Group 4 - Drug treatment started End experiment, remove tissues of interest for histological analysis

8 Steps in metastatic process Intravasation Efficiency Inefficient Is efficiency related to metastatic ability? Yes Survival in the circulation Arrest in organ Efficient Efficient No No Extravasation Efficient No Survival of cells after extravasation Initial growth of cells after extravasation Persistence of growth Inefficient Inefficient Inefficient Yes Yes Yes Breast Cancer Research (2000) 2:400-07

9 Metastatic Colonization The outgrowth of tumor cells at a distant (foreign) site Invasion Intravasation Arrest Extravasation Metastatic Colonization A Therapeutic Target? OPEN FOR THERAPEUTIC BLOCKADE Nature Cancer Rev (2003) 3:1

10 Consider the metastatic process in a breast cancer patient who is lymph node-positive at the time of diagnosis Where is the window of opportunity? Invasion Intravasation Arrest Extravasation Metastatic Colonization COMPLETED MAY OR MAY NOT BE COMPLETED OPEN FOR THERAPEUTIC BLOCKADE Nature Cancer Rev (2003) 3:1

11 Is Growth in the Primary Tumor Site the Same as the Colonization of a Distant Site? NO!!!!! The Seed and Soil Hypothesis When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil. Stephen Paget The Lancet, 1889 The soil is different The Microenvironment Primary tumor microenvironment that can facilitate or suppress invasion Metastatic site microenvironment that can facilitate or suppress colonization

12 Is Growth in the Primary Tumor Site the Same as the Colonization of a Distant Site? Metastasis suppressor genes An example that the biology is not the same Potential targets specific for metastatic disease Therapeutics When tested, primary tumors and metastases do not always respond identically to therapeutics In fact, some therapeutics may exacerbate metastasis Drug development MUST take metastasis into consideration!

13 How are drugs preclinically tested? Compound Vehicle

14 The Clinical Trial Pathway Phase I Phase II Phase III Dose escalation in metastatic patients Maximum tolerated dose Side effect profile Activity in metastatic setting Shrink established mets (CR, PR) Stabilize them (SD) Combinations with standard chemo, new agents Randomized comparison to standard of care PFS, OS Phase III Adjuvant Setting LN+ or LABC patients PFS, OS endpoints THE ONLY DRUGS EVER TESTED FOR METASTASIS PREVENTION HAVE TO WORK IN ALL THESE OTHER TRIALS.

15 SO In order to clinically test a drug for metastasis preventive activity, It has to shrink established metastatic tumors! DOES THIS MAKE SENSE????

16 How can we change this? Metastasis Research Drug Development Clinical Trial Design

17 A Success Story: Denosumab Osteoblastic metastases: Osteoblasts build bone Here they build weak, unorganized bone that breaks Osteoclastic metastases: Osteoclasts break down bone. Here they get activated and chew a hole in the bone

18 Intelligent Experimental Therapeutics : Preclinical data showed that denosumab would prevent the formation of a bone metastasis; it would not melt an already established metastasis. Trial design took this into consideration: Women with a bone metastasis randomized to standard of care (bisphosphonates) or Denosumab Endpoints: Early testing- urine markers of bone loss Phase II time to development of a new bone metastasis Time to first on-study skeletal-related event J. Clin. Oncol 25: 4431, 2007

19 A Research Example Metastasis Suppressor Genes (MSGs) Genes that mediate metastasis without affecting the primary tumor Nm23 was the first Metastasis Suppressor Gene identified Patricia Steeg, JNCI (1988)80: MSGs have been identified to date No Metastases Nm23-H1 present Metastases Loss of Nm23-H1 protein

20 The Nm23 Metastasis Suppressor Gene Metastatic Tumor Cell Vector Only Vector + Nm23 No Nm23 +Nm23 Vector + Nm23 Primary Tumor Growth: Lung Metastasis: Vector + Nm23 Vector + Nm23

21 Overexpression of Nm23-H1 Suppresses Metastasis with No Effect on Primary Tumor Size MTLN3 rat mammary adenocarcinoma cell line: Transfectant: Primary Tumor Size: Lung Metastasis: Control C % Nm23-H1 a % MDA-MB-231 Breast Carcinoma Cell Line: Transfectant: Primary Tumor Size: Lung Metastasis: Control % Nm23-H1 Cl % MDA-MB-435 Carcinoma Cell Line: Transfectant: Primary Tumor Size: Lung and LN Metastasis: Control C % Nm23-H % Int. J. Cancer 65: 531, 1996; Oncogene 8: 2325, 1993; Br. J. Cancer 78:710, 1998

22 Expression How To Bring Suppressor Genes From the Bench To the Clinic Compounds that elevate suppressor gene expression High dose medroxyprogesterone acetate for triple-negative breast cancer K. Miller, Indiana University, PI Gene therapy vectors Druggable Inverse Correlates of Suppressor Expression ET-1 for RhoGDI2 metastasis suppressor Plk1 for p53 LPA1 (EDG2) for Nm23? Aggressiveness Nm23

23 Nm23-M1 wild-type Animal Model Nm23-M1 knockout LPA1 Stained Liver Tumors Nm23-H1: Human Infiltrating Ductal Breast Carcinomas Low High LPA1: High Low Cancer Res. (2007) 67:7238 and 11751

24 Lysophosphatidic Acid Receptor (LPA1) Is An Inverse Correlate of Nm23 Metastasis Suppressor LPA is an extracellular phospholipid. It is maintained at high levels in the bloodstream (approximately 0.1 to 0.5 um) LPA is a potent motogen for tumor cells. LPA1 (or EDG2) is a G protein-coupled cell surface receptor for LPA. It transduces signals from LPA inside the cell to make it move.

25 Can LPA1 Restore Motility and Metastasis in Nm23-H1 Suppressed Tumor Cells? nm23 nm23 + LPA1 No nm23 Cell Line: Percentage of mice with metastases: No nm23: 89.3% nm23: 57.7% 46.2% LPA1: 85.7% 95.8% P value: (clonal combinations) Cancer Res. 67:7238, 2007

26 Hypothesis Inhibiting LPA1 will decrease metastasis formation in experimental model systems, acting like a metastasis suppressor gene.

27 LPA1 Inhibitor In Vivo Study Design Preclinical Studies with Debio Pharmaceutical R Day: Inject Randomize Remove Necropsy 4T1 Drug Primary PK Mfp vs. Vehicle Tumor Metastasis counts PD Markers 0719 was administered IP at a dose of 15mg/kg twice daily Experiments were performed in duplicate in 2 model systems

28 Primary Tumor Size Was Unaffected by the LPA1 Inhibitor N = 20 mice per arm P-value: NS

29 The LPA1 Inhibitor suppressed the growth of metastases in both the liver and the lungs Vehicle Debio 0719 Vehicle 0719

30 Histological Analysis of Liver and Lung Metastasis Liver P < Lung P < Vehicle Inhibitor Nm23-H1

31 Validation in a Human Breast Cancer Cell Line (MDA-MB-231) P = NS P < Vehicle 0719

32 Ki67 Ratio (Ki67/Dapi) Vehicle LPA1 Inhibitor has no effect on proliferation in primary tumors 1.6 Proliferative Index Ki67 Staining Ki67 Ratio of LPA1 Treated in Primary Tumors Control Treated Nm23-M1 Dapi Ki67

33 Percent of Ki67 positive cells Vehicle Reduced proliferation In liver metastases (also in lung metastases) Proliferative Percent of Ki67 positive Index cells in Ki67 liver metastasis Staining Control Treated P = < Dapi Ki67

34 A New Type of Drug on the Horizon: An inhibitor that acts as a metastasis suppressor gene Can Metastatic Dormancy Be Induced? What is Dormancy?

35 What is Metastatic Dormancy?

36 What is Metastatic Dormancy? phospho-p38 phospho-erk ERK (MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38 Aguirre-Ghiso et al. Cancer Research (2003) 63:1684

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38 What happens if we stop drug treatment? Dormancy is not permanent

39 In Summary LPA1 and Nm23 are inversely expressed in cell lines and tumors An LPA1 inhibitor (Debio 0719) has no effect on primary tumor growth Significant reduction in the formation of lung, liver, lymph node, spleen and other metastases Debio 0719 induced dormancy in the tumor cells that colonized the lungs and livers Site specific signaling was evident at metastatic sites including reduced Erk activation and increased p38 activation.

40 Take Home Message 1. Compounds with selective activity towards metastases exist 2. This type of compound would have been missed by traditional drug development schemes 3. Candidate drugs must be tested in metastatic models and clinical trials designed to question efficacy in the metastatic setting

41 How do we test agents for prevention of metastasis the clinic? Identify women at high risk for development of metastases - Lymph node positive at time of primary diagnosis - Locally advanced primary disease - For site specific metastasis, diagnosis of a - Ultimately with gene expression profiling Trial Design: Secondary Prevention Randomized phase II trials to administer standard of care chemotherapy +/- potential preventive agent Primary Endpoint: Time to development of a new metastasis Additional important information: ADVERSE EFFECTS, COMPATIBILITYWITH CHEMOTHERAPY, QUALITY OF LIFE

42 Perspective: The Right Trials (2012) Steeg, P. Nature, 485:S58

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44 Laboratory of Dr. Patricia Steeg Women s Cancers Section, NCI Jean-Claude Marshall, Joji Nakayama, Natascia Marino, Joshua Collins Diane Palmieri NCI: Seth Steinberg, Statistical Analysis Mary Albaugh, Laboratory Animal Sciences Debio Pharmaceuticals: Maximillian Murone Maryse Barbier JC Marshall Spain: Fernando Vidal-Vanaclocha, Madrid