The Expression of the Ubiquitin Ligase Subunit Cks1 in Human Breast Cancer Merav Slotky, Maanit Shap

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The Expression of the Ubiquitin Ligase Subunit
Cks1 in Human Breast Cancer Merav Slotky,
Maanit Shapira, Ofer Ben-Izhak, Shai Linn, Boris
Futerman, Medy Tsalic and Dan D Hershko (2005)
Presented by Julie Schuck
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Why investigate gene expression in tumors?

• To help physicians select the best therapy for
  each patient. A therapeutic intervention may be
  more effective on some cancer types than on
  others.
• To improve accuracy in predicting disease
  outcome. Prognosis through traditional factors is
  not very accurate.

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Genes investigated in this study

• p27Kip1
• Skp2
• Cks1

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p27Kip1 Cyclin-dependent kinase inhibitor

• Down-regulation is associated with poor prognosis
  for many cancers
• Decrease in p27Kip1 results from rapid
  degradation by the ubiquitin-proteasome pathway

RV Lloyd et al., 1999
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Skp2 S-phase kinase protein 2

• Part of a ubiquitin ligase complex
• Skp2 specifically binds p27Kip1 and targets it
  for degradation by the ubiquitin proteolytic
  system

http//www.bris.ac.uk/Depts/BHI/mark/ecm.html
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• Cks1 Cyclin dependent kinase subunit 1
• An essential cofactor for Skp2-dependent
  ubiquitylation of p27Kip1

(Bartek and Lukas, 2001)
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Questions investigated in this study

• What relationship does Cks1 expression have with
  Skp2, and p27Kip1 in breast cancer?
• What relationship does Cks1 expression have with
  traditional diagnostic factors?
• What is the relationship between Cks1 expression
  and disease-free and overall survival?

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Materials and Methods

• Samples were obtained from two sources
• 1. Tissue samples of 50 primary breast
  carcinoma patients (collected more than 6 years
  previous to the study).

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• 2. Cell cultures from 3 different human breast
  cancer cell lines MCF7, T47D, and MDA-MB-231

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Immunoblotting

• Separate proteins by gel electrophoresis
• Transfer proteins to a nitrocellulose membrane
• Probe with antibodies to Skp2, Cks1, p27Kip1, and
  Skp1 (control)
• Visualize the proteins with horseradish-conjugate
  d IgG and chemoluminescence
• All immunoblot analyses were performed at least
  twice.

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Gel Electrophoresis
http//www.bergen.org
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Nitrocellulose membrane
http//www.labfilters.com
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• Staining with Antibodies
• Skp2
• Skp1
• p27Kip1
• Cks1

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Preparing the tissue for antibody staining

• Deparaffinize with xylene
• Rehydrate in a series of ethanols
• Process in Antigen retrieval processor or
  microwave, with ethylenediaminetetraacetic acid
  buffer.

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Antibody staining

• Skp2 Diluted to 1100
• Stained in the NexES IHC immunocontainer
• Cks1Diluted to 140
• Incubate 2 hrs at room temp.
• Completed with the Super sensitive Multilink
  Immunodetection kit
• P27Kip1Diluted to 1500
• Incubated overnight at 4 C, finished with
  Histostain plus kit
• Negative controls were treated with 10 nonimmune
  rabbit serum.

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• Scoring gene expression
• Criteria percentage of cells showing nuclear
  staining
• p27Kip1-- lt50 low expression, gt50 high
  expression
• Skp2-- lt10 low expression, gt10 high
  expression
• Cks1 -- lt10 low expression, gt10 high
  expression

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Statistical Analysis

• Relationship between the various proteins
  Pearsons X2 and cross-tabulation
• Relationship between the proteins and clinical/
  pathological features of the samples Pearsons
  X2 and cross-tabulation
• Survival curves Kaplan Meier method, Cox
  regression
• P values less than 0.05 were considered
  significant.

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Cks1 levels in breast cancer (in tissue samples)

• Low or absent in adjacent normal tissue
• Variable levels in tumor specimens
• Direct relationship with Skp2 (r 0.477,
  p0.001)
• Inverse relationship with p27Kip1 (r -0.726,
  plt0.001)

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A strong inverse relationship between p27Kip1 and
Skp2 / Cks1 was found in 74 of the tissue
samples

• High levels of Cks1 and Skp2 (h) accompanied low
  levels of p27Kip1(i) in 15 patients
• Low levels of Cks1 and Skp2 (i) were observed
  with high levels of p27Kip1 (h) in 22 patients.
• 13 out of 50 patients did not show this inverse
  relationship.

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Cks1 and traditional diagnostic factors

• Cks1 over-expression was associated with
• Decreased tumor differentiation (r-0.421,
  p0.012)
• Young age (r0.327, p0.049)
• Estrogen Receptor negative status (r -0.356,
  p0.013)
• Progesterone Receptor negative status (r -0.404,
  p0.006)
• --but not lymph node status and tumor size

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Protein expression in cell lines

• Levels of Ck1 and Skp2 were higher in
  estrogen-independent cell lines.
• Levels of p27Kip1 were lower in
  estrogen-independent cells

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• Cks1 and Skp2 levels were higher T47D cells than
  MCF7.
• Estradiol increased Cks1 and Skp2 levels in T47D
  cells.
• Tamoxifen downregulated Cks1 and upregulated p27
  in T47D cells.

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Cks1 levels and patient survival

• 80-month follow up data was analyzed for all 50
  patients.
• Cks1 over-expression in tumor samples was
  associated with
• poor disease-free survival
• poor overall survival

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• Overall recurrence of breast cancer
• Local recurrence
• Distant recurrence

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Survival
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Conclusions

• 1. Cks1 protein expression is directly related to
  Skp2 expression and inversely related to p27Kip1
  expression in human breast cancer
• 2. Cks1 overexpression is associated with poor
  tumor differentiation, young age, and negative
  ER/PR status
• 3. Cks1 overexpression is associated with poor
  disease-free and overall survival in human breast
  cancer

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• Cks1 may serve as
• A prognostic marker
• A target for intervention

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• Possible intervention-- siRNA