Regulation of Breast Cancer Cell Chemotaxis by the Phosphoinositide 3-Kinase p110

1. Regulation of Breast Cancer Cell Chemotaxis by the Phosphoinositide 3-Kinase p110 Carol Sawyer, Justin Sturge, Dorothy C. Bennett, Michael J. O’Hare, William E. Allen, Jennifer Bain, Gareth E. Jones and Bart Vanhaesebroeck

2. Every twelve minutes a woman in America dies of breast cancer This year alone, more than 211,000 women in the U.S. will be diagnosed with the disease 43,300 of women diagnosed with breast cancer will die this year One woman in eight either has or will develop breast cancer in her lifetime

3. What is breast cancer? Breast cancer is the uncontrolled growth of malignant breast tissue How is breast cancer detected? • Breast self examination • Mammography Mammography is a special X-ray technique that is used to visualize soft tissues of the breast as a means for screening women for breast cancer. e.g. of a Mammogram

4. Staging breast cancer • The development of breast cancer is categorized • into eight separate stages • stages 0, I, IIA, IIB, IIC, • IIIA, IIIB, IIIC • and IV

5. Breast Cancer Treatment • Surgery • lumpectomy • mastectomy • biopsy • Radiationtherapy • Chemotherapy • Hormone therapy • estrogen inhibition

6. Regulation of Breast Cancer Cell Chemotaxis by the Phosphoinositide 3-Kinase p110 Carol Sawyer, Justin Sturge, Dorothy C. Bennett, Michael J. O’Hare, William E. Allen, Jennifer Bain, Gareth E. Jones and Bart Vanhaesebroeck

7. Phosphatidylinositol 3-kinases (PI3Ks) • Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases • There are three major classes of PI3Ks Class I, Class II and Class III • Class IA PI3Ks • comprised of a regulatory and catalytic subunit • function to generate 3-phosphoinositide lipids within cell membranes • 3-phosphoinositide lipids interact with numerous intracellular target proteins • the PI3K pathway regulates numerous cellular processes, such as proliferation, cell motility and apoptosis • deregulation PI3K-pathway has been implicated various cancers Chemical structure of phosphatidyl-inositol

8. The Role of Upstream and Downstream targets of PI3K enzymes in cancer • Tyrosine kinases and Ras are involved in signaling pathways upstream of PI3K are generally overexpressed or mutationally activated in cancer • Akt/protein kinase B acts downstream of PI3K to regulate many biological processes such as proliferation, apoptosis and growth

9. The Role of Upstream and Downstream targets of PI3K enzymes in cancer(cont’d) • Overexpression of Akt/protein kinase B a downstream target of PI3K has been implicated in gastric, ovarian, prostate and breast tumors • The tumor suppressor protein PTEN dephosphorylates 3-phosphoinositides, products of PI3Ks • PTEN inactivation leads to an accumulation of PI3K lipid products and consequently up-regulation of the many PI3K-regulated cellular activities

10. Structure of Class IA PI3Ks Class IA PI3Ks is an heterodimeric enzymes which consist of an SH2 -domain-containing regulatory subunit in complex with a p110 catalytic subunit Structure of PI3K

11. Activation of Class IA PI3Ks Class IA PI3Ks are activated via the direct interaction of Ras (p21ras) with the and/or the interaction of the regulatory subunit with phosphorylated tyrosines

12. The Different Isoforms of PI3K • Regulatory subunits: p85α, p85β and p55γ • Catalytic subunits: p110α, p110βand p110δ • p110α and p110β are widely distributed in mammalian tissues • p110δ is mainly found in leukocytes • All p110 isoforms are identical in regards to activation processes such as regulatory subunit interaction, recruitment by phosphotyrosine complexes and lipid substrate specificity • Isoforms p110 differ in protein kinase activities, interaction with Ras and regulation of lipid kinase activities

13. The tissue distribution of p110δ mRNA based on EST analysis • In order to gain their own insight into the distribution of class IA PI3Ks Sawyer et al. retrieved ESTs for each PI3K isoform from the GenBank and categorized them into various tissue groups. • Similar total numbers were collected for each p110 isoform. • The largest proportions of p110δ were found in blood/immune cells. • There is a broader tissue distribution of p110α and p110β mRNAs compared to relatively limited distribution of p110δ. Fig1a.

14. Analysis of p110δ m RNA distribution excluding contamination from WBC • Relatively weak p110 response can be seen in nonleukocyte cell lines while a stronger signals is seen in the melanoma cell line. • The response was greatest in the cell lines derived from WBC origins. Fig 1b

15. Are PI3K mRNA levels a true representation of PI3K protein expression? • cell lysates were analyzed for the expression of p110 proteins by immunoblotting using antibodies specific for the various p110α, p110β and p110δ isoforms. • Both the normal breast cells and primary tumor breast cells expressed all classes of PI3K isoforms. • The levels of p110α in the tumor cells were frequently low Fig 2a

16. Analysis of different breast cancer cell lines for the expression of p110 isoforms • p110α and p110β isoforms were expressed in all cell lines • 9 of the 15 cell lines showed expression of the p110δ isoform • no correlation between with p110δ expression and a specific tissue origin could be demonstrated • The reason for the variation in p110δ expression in different cell types is unknown. The variation in expression however indicates that the expression of p110δ is not always necessary for in vitro cell propagation Fig 2b

17. Role of PI3K in Breast Cancer Cell Migration • MDA-MB-231 breast cancer cells • express p110β, p110δ and p110α at levels similar to WBCs. • respond to EGF by intracellular PI3K activation • heterogenous in size and shape • After treating the cells with EGF for 2 min (Short-term treatment) there was extensive membrane ruffling and a reduction in the number of actin stress fibers and the cells also adopted a polarized morphology • After incubation of the cells with LY294002, a PI3K inhibitor which inhibits all of the class IA PI3K isoforms, EGF-induced membrane ruffles were completely blocked, there was no loss of stress fibers, and the creation of a stable leading edge was inhibited Fig 4

18. The relationship between PI3K activity and the directional cell migration • LY294002 treatment: • inhibits an increase in cell movement (Fig. 5B), • prevented directional migration, and had a slight inhibitory effect on cell speed (Fig. 5C) (Sawyer et al., 2003) • Therefore PI3K must be required for EGF-induced MDA-MB-231 cell motility Fig 5c Fig 5b

19. The specific role of p110δ isoform in directional cell migration • Only the antibodies to p110δ and not p110α p110β were found to inhibit cell movement in any one direction • p110δ is required by breast cancer cell lines to provide a directional response to a chemoattractive stimulus Fig 6b Fig 6a

20. Critical points pertaining to PI3Ks • PI3Ks are critical effectors of signaling pathways of tyrosine kinase and Ras which are often deregulated in various cancers. • All mammals possess three different PI3K p110 (the catalytic subunit) isoforms, p110α, p110β and p110δ. • p110α and p110β were detected in every tissue and cell line investigated. The expression of p110δ on the other hand was much more limited. • p110δ can regulate cell migration in breast cancer cell lines. • Treatment with the broad-spectrum inhibitor LY294002 has the same impact on cell migration as does incubation with p110δ alone. • p110δ is the most important p110 isoform for the regulation of in vitro chemotactic migration in response to EGF and provides support for a biological function of p110 in breastcancer cells

21. Conclusions drawn from the research of Sawyer et al. • This study provides evidence for a role of chemotaxis in the regulation of breastcancer and melanoma metastasis, as these types of tumors share a similar pattern of metastatic sites, namely the lymph nodes, lung, liver, and bone marrow . • The expression of p110 in breast cells and melanocytes may be somewhat related to their similar migratory activities under normal and tumorgenic conditions . • Precisely how PI3Ks or the different p110 isoforms regulate chemotaxis and the organization of the actin cytoskeleton is not known; however, Sawyer et al. hypothesize that it is likely to involve a polar distribution of PI3K lipid products after cell stimulation . • At present p110 is considered to be a prime target for anti-inflammatory drugs due to in a large part because of its prevalence in WBCs. • p110 is now considered to be a potential target for antimetastatic agents

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