Dean’s Undergraduate Research Opportunities Program (DUROP)
Project Description – Summer 2015
Project Title
Testing the impact of LKB1 loss-of-function in an experimental 3D model of ovarian
cancer metastasis
Supervisor: Dr. Trevor Shepherd
Department: Obstetrics & Gynaecology; Oncology; Anatomy & Cell Biology
Project Description
Background: Ovarian cancer is the most lethal of the gynaecologic cancers in the developed world
principally due to its late-stage diagnosis and high rate of recurrence of chemotherapy-resistant disease.
Ovarian cancer is unique among solid tumours in that it metastasizes, or spreads, by direct tumour cell
dissemination into the peritoneal cavity. Ovarian cancer cells commonly cluster to form three-dimensional
spheroids, which enhance cell survival, metastatic potential and resistance to chemotherapy. We have
recently discovered that ovarian cancer cells in spheroids upregulate the metabolic stress response
mediator Liver kinase B1 (LKB1). The gene encoding LKB1, STK11, is thought to be a tumour suppressor
in several malignancies, including some ovarian cancers, yet our results support an alternative and
necessary role for this metabolic stress kinase in supporting cell survival in 3D spheroids, and
potentially to promote metastasis.
Hypothesis: LKB1 function is required for ovarian cancer cell viability in 3D spheroids.
Methodology: Ovarian cancer cell lines HeyA8 and OVCAR8 will be engineered to lack a functional
STK11 gene (encoding LKB1) through the use of Cas9-mediated genome editing technology. (11 clones
have already been generated for HeyA8 cells, and OVCAR8 clones are in progress.) Loss of LKB1
expression and function will be validated by western blotting of protein extracts isolated from STK11knockout clones as compared with parental HeyA8 and OVCAR8 cells. Several clones of each will be
tested for: (1) adherent cell proliferation and viability by alamarBlue assay; (2) spheroid formation by
seeding cells in suspension to Ultra-Low Attachment plates and documenting size, number and assaying
for cell viability using CellTiter-Glo; (3) spheroid reattachment and quantifying spheroid attachment and
cell dispersion; and (4) adherent cell and spheroid cell viability after treatment with the ovarian cancer
chemotherapeutic drug carboplatin. All assays are routinely performed by our laboratory and therefore no
technical obstacles are expected. If time permits, protein lysates will be generated from parental cell lines
and STK11-knockout clones for reverse phase protein array analysis (services accessed through MD
Anderson Cancer Center) of intracellular signalling proteins to identify the substrates and pathways that
LKB1 regulates in ovarian cancer cells and spheroids to promote cell viability and metastatic potential.
Expected Outcomes: We predict that loss of LKB1 function in ovarian cancer cells will negatively impact
cell survival in 3D spheroids, yet should not affect the viability of proliferating, metabolically-active
adherent cells. In fact, loss of LKB1 function may re-sensitize ovarian cancer cells to carboplatin within
chemo-resistant spheroids.
Schulich School of Medicine & Dentistry, Western University
Rix Clinical Skills Bldg, London, ON, Canada N6A 5C1
t. 519.661.2111 f. 519.850.2357 www.schulich.uwo.ca
Research Environment
The summer student will perform the project in the basic research lab of the Translational Ovarian Cancer
Research Program (TOCRP) at the LRCP. There are currently 2 PhD students from Anatomy & Cell
Biology and a full-time research technician. The student will be directly supervised by Dr. Shepherd and
co-supervised by Dr. Gabriel DiMattia (Oncology, Biochemistry), who is co- lead scientist of the TOCRP
with Dr. Shepherd. The student will be trained to perform all experiments by the research technician or Dr.
Shepherd directly. The laboratory has bench space for up to 8 trainees and all equipment that is required
to perform experiments (e.g. laminar flow hoods, incubators, microscopes, plate reader, etc.) are
available in the lab or as part of the core facilities of the Cancer Research Laboratory Program at the
LRCP.
Expected Objectives/Accomplishments for Student within 16 weeks
The student will be expected to learn the essential skills of standard mammalian cell culture, perform cell
viability assays, acquire digital images of cells and 3D spheroids by light microscopy and perform western
blotting of protein extracts from cells/spheroids. Interpretation and presentation of results by the student
will be done one-on-one with Dr. Shepherd and at weekly lab meetings. The student will be expected to
attend the Department of Oncology Research & Education Day in June.
Dr. Shepherd: tshephe6@uwo.ca
519-685-8500 x56347
LRCP A4-836