# Presentation: 948958

## Positive and Negative Regulators of Metastasis

**Positive and Negative Regulators of Metastasis**

**J. Carl Barrett**
**Laboratory of Biosystems and Cancer**
**Center for Cancer Research**
**National Cancer Institute**

## Slide 2

## The Metastatic Cascade

**The Metastatic Cascade**

## When does metastasis begin?

**Commitment to the metastatic phenotype:**
**How early does it occur?**
**Can it be reversed?**
**Progenitor lesions:**
**What are the key progenitor lesions?**
**What is the efficiency of transition to invasion?**
**Are all metastasis precursors clonal?**

## What is the role of the host?

**Under what conditions does the host drive or suppress the process?**
** ****Does the transition from pre-invasive to invasive lesions require host participation?**
**If so what are the molecular and cellular players that are functionally important?**
**The circuitry of the tumor host communication may be the key to prevention of invasion.**

## Physiologic basis of metastasis

**Is metastasis a normal physiologic program which is disregulated or inappropriately activated?**
**Does a physiologic motility and invasion program exist for development, angiogenesis morphogenesis and wound healing?**
**Is metastasis colony formation a natural ongoing process conducted by stem cells? **

## What is the driving force?

**Is the metastatic phenotype pre determined within the primary tumor? Within the host microenvironment?**
**Are malignant cells a product of adaptation and selection? **
**What is the selection factor? If malignant cells are survival of the fittest, then what is the fitness test?**
**Is cell survival in a foreign (non home) tissue the ultimate selection factor? **

## Metastasis Pre-1900

- Pre-cellular theory of  invasion and metastasis: recognition of malignant tumors and localized versus metastatic disease

**LeDran 1757: **Noted that malignant tumors begin as localized disease, then spread to regional lymph nodes and then enter the circulation to subsequently appear in the lung

**Bichat 1801: **Tumors contain both parenchyma and stroma
**Recamier 1829 :** Used the term “Metastases”

**Metastasis Pre-1900**

## Validation of the cellular theory of cancer metastasis

**Validation of the cellular theory of cancer metastasis**

**Tyzzer 1913:** Experimental Metastasis

**Ziedman and Fidler 1970-80:** Intravenous metastasis models

##       The organ pattern of metastasis is characteristic of the tumor type and tissue of origin. 50-70% of the metastatic pattern can be predicted by the venous drainage blood flow. The remaining 30-50 % may be caused by specific molecular homing mechanisms.

- Potential molecular mechanisms:
- Preferential adhesion in the vessels of the target organ
- Selective extravasation
- Organ attractants
- Organ specific survival and growth

-       The organ pattern of metastasis is characteristic of the tumor type and tissue of origin. 50-70% of the metastatic pattern can be predicted by the venous drainage blood flow. The remaining 30-50 % may be caused by specific molecular homing mechanisms.

## Chemokines regulate leukocyte recirculation and trafficking to sites of inflammation and infection

## Premise:

- Metastasis homing is dictated by relative abundance of  chemokines and cognate receptors on the tumor cell.

- Why do the tumor cells express the chemokine receptors in the primary tumor prior to dissemination?

- Therapeutic utility is limited because dissemination has already occurred at the time of diagnosis

## Hypoxia induces angiogenesis and promotes invasion

**Hypoxia induces angiogenesis and promotes invasion**

- Outgrowth of vascular supply

- Selection of aggressive cells

**Tumor necrosis is a bad prognostic indicator**

## Translational Applications

**Translational Applications**
- Extracellular signalling networks are the therapeutic target
- Pro survival pathways may be a key selection factor within a given cellular context

**Molecular Ecology of the Tumor-Host Microenvironment**

**Pathogenic defects in signal pathways extend into the tumor host interface****.**** **
-  Cross regulation, and exchange, of enzymes, substrates, cytokines and motogens stimulates motility, invasion, survival and growth of tumor and host populations
-   Locally and tightly regulated at the leading edge of the invading cancer cell

## Key Theme

**   ****As with the other stages of carcinogenesis, metastasis is genetically controlled with the involvement of both enhancing and suppressing modifiers.**

## Metastasis Promoting Genes - I

| Gene | Tissue Site | Function |
| --- | --- | --- |
| ARM-1 | Lymphoma | Promotes adhesion of tumor cells to the endothelium |
| ATX | Breast, Liver, Lung, Melanoma, Teratocarcinoma | cytoskeletal reorganization and motility; G-protein coupled receptor activation |
| CD44 | Multiple sites | cell-cell interactions; activates HGF/c-Met pathway |
| Cox2 | Breast, Colorectal, Gastric | Prostaglandin synthase; induces VEGF |
| Cyr61 | Breast | Mediates adhesion; Erb-B2/3/4 pathway |
| Ezrin | Liver, Ovary, Pancreas, Prostate, Uterus | Membrane-cytoskeletal linker; RHO and RAC interactions |
| HMG-I(Y) | Breast, Cervical, Colorectal, Prostate, Skin, Thyroid, Uterus | Regulated by EGF and MMP-9 |
| Laminin-5 | Multiple sites | EGF and TGF-induce expression of laminin subunits; cell adhesion, motility |
| c-Met | Multiple sites | Activated by HGF; Modulates Ras and PI3 kinase |

## Metastasis Promoting Genes - II

| Gene | Tissue Site | Function |
| --- | --- | --- |
| MTA1 | Breast, Cervix, Melanoma, Ovary | Neucleosome remodeling; histone deacetylase complex |
| Oncostatin M | Lung | Activates PKA-dependent pathway |
| PP2A | Not determined | Activated by p38/MAPK; inhibits MEK1, MEK2, and MMP-1 |
| RAGE | Gastric, Lung, Pancreatic, Renal | transmembrane receptor; activates p21, MAPKs, NF-6B, cdc42/rac |
| S100A4 | Breast, Colorectal, Prostate | Calcium-binding protein; activates c-erbB-2 |
| S100A9 | Colon, Gastric, Skin | Calcium-binding protein; Modulates Mac-1 integrin receptor through G-protein |
| Semaphorins | Gastric, Leukemia, Lung, Skin | cell-cell interactions; Receptor crosstalk with c-Met binding semaphorin receptor, plexin  |
| Thymosin-15 | Prostate | actin binding; motility |
| Wnt-5a | Breast, Colon, Lung, Melanoma, Pancreas, Prostate | PKC activation with associated changes in cytoskeleton, cell adhesion, and motility |

## Cellular Phenotypes Modulated by IGF1

- Growth
- Apoptosis
- Invasion
- Metastasis
- Angiogenesis
- Response to chemotherapy

## IGF-1/IGF-R as Positive Regulators of Metastasis

- Mutant IGF-R(soluble receptor) blocks metastasis but not tumor growth of breast cancer cells(Dunn/Barrett).
- Serum IGF-1 levels influence metastasis of colon cells(Wu/LeRoith).
- IGF-R overexpression accelerates metastatic progression in RIP-Tag mice(Lopez/Hanahan).

## Evidence for Genetic Influences on Metastatic Potential

- Metastasis formation(independent of tumor initiation and growth) in mice is dependent on the genetic background of the mouse and map to multiple loci (Kent Hunter, NCI)
- Hybrids between metastatic cells and non metastatic cells are suppressed for metastasis independent of tumor forming ability
- Specific genes can control metastasis independent of tumorigenesis

## Metastasis Suppressor Genes - I

| Gene | Tissue Site | Function |
| --- | --- | --- |
| Annexin7 | Prostate | calcium-dependent GTPase; substrate for PKC and other kinases associated with proliferation |
| BRMS1 | Breast, Melanoma | gap-junctional communication |
| CC3 | Colon, Lung | serine/threonine kinase |
| CEACAM1-4S | Breast, Colon | Bax pathway |
| CRSP3 | Melanoma | transcriptional co-activator |
| DAP-kinase | Multiple sites | calcium/calmodulin-dependent serine/threonine kinase; pro-apoptotic pathway |
| E-cadherin | Multiple sites | Wnt signaling; cytoskeleton; cell-cell adhesion |
| HEPSIN | Ovarian, Prostate, Renal | transmembrane serine protease |
| HPIHS | Breast | non-histone heterochromatin-associated protein |
| KAI-1 | Breast, Prostate | Transmembrane tetraspondin; role in adhesion, motility, growth regulation, and differentiation; integrin interaction |
| KiSS1 | Breast, Melanoma | Modulates Rho, Rac, and MAPK signaling  |
| Maspin | Breast, Colon, Oral Squamous Cell, Prostate | Serine protease inhibitor; binds collagen and can modulate integrins |
| Melastatin | Melanoma | Calcium channel protein |

## Metastasis Suppressor Genes - II

| Gene | Tissue Site | Function |
| --- | --- | --- |
| MKK4 | Ovary, Prostate | MAPK; phosphorylates and activates p38 and JNK kinases |
| NESH | Lung, Prostate | src homology 3 adapter protein; down regulates p21 pathway |
| NM23-H1 | Breast, Colon, Melanoma, Oral Squamous Cell | histidine kinase; phosphorylates KSR, which might reduce ERK 1/2 activation |
| PTEN | Multiple sites | phosphatase; growth regulation, cell motility |
| RhoGD12 | Bladder | Inhibits GTP binding; regulates RHO and RAC |
| SFRP1 | Breast, Colorectal | Modulates Wnt signaling pathway |
| SHPS-1 | Breast, Leukemia | glycoprotein; may regulate RAS-MAPK signaling; suppresses anchorage independent growth |
| Syk | Breast, Colon, Pancreas, Skin | Tyrosine kinase; inhibits PI3 kinase; necessary for MAPK activation |
| TSP-1 | Multiple sites | inhibits endothelial cell proliferation and migration; c-Myc expression inhibits TSP-1 |
| tropomyosins | Breast | interacts with e-cadherin/catenin complex |
| VDUP1 | Melanoma | Thioredoxin inhibitor; upregulates KiSS1; interacts with CRSPs |

## Lessons Learned from Studies of Genes Involved in Metastasis

- Both positive and negative regulation of metastasis are involved in cancers.
- Metastasis suppressor genes can be lost early in the development of cancers.
- Multiple mechanisms are involved in metastasis.
- Interactions and possible pathways of proteins involved in metastasis are observed.
- Negative regulators of metastasis often exhibit epigenetic silencing rather than mutations in cancers.
- Negative regulators of metastasis exhibit plasitcity of expression and function .

## Prostatic Metastasis Suppressor Gene

- KAI-1

## Slide 25

**Notes:**
11d

## KAI1 / CD 82

**KAI1 / CD 82**

**Names :**** KAI1 / CD82, (C33, R2, IA4)**

**Gender :**** ****Transmembrane Glycoprotein**

**Member of the tetraspanin or transmembrane 4 superfamily (TM4SF)**

**Contains an internalization sequence at its C-terminus (YSKV)**

***Current Address***** :**

**Cell membrane (lymphocytes, epithelial cells)**

**Lysosomes**

**Vesicles**

**Ligands**** ****?**

**Biological Function**** :**
**	****motility**
**	****invasiveness**
**	****cell-cell interactions**

***Particularity***

****

****

**Signal Pathways**** : ****?**

## KAI1 / CD 82 and Cancer

**prostate****	****	****lung**
**pancreas****	****carcinoma**
**colon**

**prostate**
**lung carcinoma**
**colon**
**hepatoma**
**breast**
**lung (non-small-cell carcinoma)**
**bladder  cancer**
***ovary***
***melanoma***

**KAI1/CD82 expression is inversely related to the metastatic potential :**

**Transfection of tetraspanin reduces metastatic potential**

**melanoma**
**prostate**
**breast**

**B16**
**MDA-MB-435 *******
**AT6.1, AT6.3**
**MDA-MB-231**

**KAI1 / CD 82 and Cancer**

**Correlations**

**Experimental Data**

**(from Boucheix & Rubinstein , 2001**

## Loss of KAI-1 Expression in Prostate Cancer

**Loss of KAI-1 Expression in Prostate Cancer**

**Notes:**
#16

## Slide 29

**Notes:**
#15D
PIN
C33 KAI1 mAb

## Slide 30

**Notes:**
16d

## KAI-1 Functions

| + (Promotes) | - (Inhibits) |
| --- | --- |
| Cell Aggregability
Cell Adhesion | Invasion
Motility
Metastasis |

## The Key Question :

**The Key Question :**

## KAI-1 as a Molecular Facilitator

## Association of KAI1 / CD82 with other cell surface molecules

*** **integrins*

- EGF-R

*Molecules of the immune system*

*Others*

- DARC

- E-Cadherin

*TM4SF members*

**the tetraspanin web**

## Confocal microscopy

**Confocal microscopy**

**MCF-7**

**MCF-7**
**- ****Kai1-9**** **

**KAI1**

**E-Cadherin**

**merge**

**objective x100**

## EGF-R

**EGF-R**

**Names :**** EGF-R (erb-1)**

***Gender***** ****:**** ****Receptor Tyrosine Kinase**** (RTK)**

**Signal Pathways :**

**Ligands:****   EGF**
**	****     ****TGF******

**MAPK**
**PI3K**

**Biological Function :**
**	****morphogenesis**
**	****growth regulation**

**EGF-R pathway**

## Why KAI-1 and EGF-R pathways ?

- 

**Attenuation of EGF receptor signaling by a metastasis suppressor, the tetraspanin CD82/KAI1**

**E. Odintsova et al., 2000**

**Facilitation of ligand-induced endocytosis of the EGF-R and its subsequent desensitization by CD82/KAi1**

**Selective enrichment of Tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B- lymphocytes**

**J.M. Escola et al., 1998**

**Opposite effects of KAI1 and EGF-R pathway**

**“****Co-localisation” in endosomes of KAI1 and EGF-R**

## Possible Mechanisms for Loss of KAI-1 Function

**Mutation -never observed**
**Down regulation of mRNA -                         common in many cancers**
**DNA methylation of promoter-                             not observed**
**Posttranslational modification -                 glycosylation differences observed in some tumors**
**Loss of function in KAI-1 partners or downstream effectors -   not fully tested**

## Regulators of KAI-1 expression in cancer cells

- 5-AzadC
- Phorbol esters
- Nerve growth factor
- TNF/NFkB

## A Metastasis Suppression Pathway

- DRIP130

- 6

- GPR54

**?**

- Motility
- Invasion

- VDUP1

- KISS1

- TRX

- Transcription

- ASK1/MAPKKK5

## Metastasis Suppressor Pathways

- R
- T
- K

- C
- D
- H
- 1

- EGFR

- RAS

- CRSP3

- RAF

- MEK

- ERK

- Growth
- Differentiation

- Apoptosis
- Inflammation
- Differentiation
- Survival

- -Catenin

- K
- A
- I
- 1

- RhoGDI2

- F
- A
- S

- KISS1

- NFB

- ASK1

- MKK7

- JNK

- MKK4

- MKK6

- p38

- MKK3

- Apoptosis
- Cytokines

- Survival
- Angiogenesis

- TXNIP

- TXN

- HIF

- VAV

- KSR

- NME1

- Motility
- Invasion

- G
- P
- R
- 5
- 4

- I
- G
- F
- R
- 1

- M
- E
- T

- PI3K

- PLC

- AKT

- BAD

- PTEN

- Rho

- Rac

**Metastasis Suppressor Pathways**

## Lessons Learned from Studies of Genes Involved in Metastasis

**Both positive and negative regulation of metastasis are involved in cancers**
**Multiple mechanisms are involved in metastasis**
**Interactions and possible pathways of genes involved in metastasis are observed.**
**Negative regulators of metastasis often exhibit epigenetic silencing in cancers.**
**Negative regulators of metastasis exhibit plasticity of expression and function **

## Hard Clinical Truths About Metastasis

**Upwards of 70% of patients may have overt or occult metastases at diagnosis.**
**Acquisition of the invasive and metastatic phenotype is an early event in cancer progression.**
**Millions of tumor cells are shed daily into the circulation.**
**Angiogenesis is a ubiquitous and early event that is necessary for and promotes metastatic dissemination.**

## Lucky Clinical Truths About Metastasis

**Both malignant invasion and angiogenesis use the same “hardware” and “software” programs.**
**Less than 0.01% of circulating tumor cells successfully initiate a metastatic focus.**
**Circulating tumor cells can be detected in patients who do not develop overt metastatic disease.**
**Metastases may be as susceptible to anti-cancer therapy as primary tumors.**

## Slide 45