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

1
Regulation of Breast Cancer Cell Chemotaxis by
the Phosphoinositide 3-Kinase p110 Carol
Sawyer, Justin Sturge, Dorothy C. Bennett,
Michael J. OHare, 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

• Radiation therapy
• 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. OHare, 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(contd)

• 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 p85a, p85ß and p55?
• Catalytic subunits p110a, p110ßand p110d
• p110a and p110ß are widely distributed in
  mammalian tissues
• p110d 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 p110d 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 p110d were found in
  blood/immune cells.
• There is a broader tissue distribution of p110a
  and p110ß mRNAs compared to relatively limited
  distribution of p110d.

Fig1a.
14
Analysis of p110d 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 p110a, p110ß and p110d
  isoforms.
• Both the normal breast cells and primary tumor
  breast cells expressed all classes of PI3K
  isoforms.
• The levels of p110a in the tumor cells were
  frequently low

Fig 2a
16
Analysis of different breast cancer cell lines
for the expression of p110 isoforms

• p110a and p110ß isoforms were expressed in all
  cell lines
• 9 of the 15 cell lines showed expression of the
  p110d isoform
• no correlation between with p110d expression and
  a specific tissue origin could be demonstrated
• The reason for the variation in p110d expression
  in different cell types is unknown. The variation
  in expression however indicates that the
  expression of p110d 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 p110ß, p110d and p110a 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 p110d isoform in directional
cell migration

• Only the antibodies to p110d and not p110a p110ß
  were found to inhibit cell movement in any one
  direction
• p110d 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, p110a, p110ß
  and p110d.
• p110a and p110ß were detected in every tissue and
  cell line investigated. The expression of p110d
  on the other hand was much more limited.
• p110d 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 p110d alone.
• p110d 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 breast cancer
  cells

21
Conclusions drawn from the research of Sawyer et
al.

• This study provides evidence for a role of
  chemotaxis in the regulation of breast cancer 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

22
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