Does MNRR1 lead to the Metabolic Flexibility in Ovarian Cancer Cells?

Shawn Jayee

Problem

• Patients diagnosed with ovarian cancer have a 30% chance of survival after 5 years
• Ovarian cancer goes through metastasis early and is characterized by micrometastasis
• Micrometastasis leads to occlusion which then leads to patient mortality

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General Objective

• Understand mechanisms that promote ovarian cancer metastasis

Hypothesis

• The metabolic flexibility of ovarian cancer cells is required for the metastatic potential

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• MNRR1 is the primary regulator for metabolic flexibility of ovarian cancer cells

MNRR1

• Protein
• Gene regulation
• Anti apoptosis
• Improvement of mitochondrial respiration/ ATP production

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*These functions depend on the cellular location of MNRR1 and what proteins it is bound to

Specific Objective

• Where is MNRR1 found in different ovarian cancer cells?
• In the nucleus or the mitochondria, or both?
• What is MNRR1 bound to in different ovarian cancer cells?
• Does binding to certain proteins increase the metabolic flexibility of ovarian cancer cells.

Method Number 1

• What is fractionation?
• A process used to separate the cellular components in the cell
• Because the cells are lysed, it separates the proteins contained in the nucleus from the proteins contained the in cytoplasm (mitochondria)
• Experimental design (first): Fractionation
1. Fractionation: To find where the MNRR1 is found, the different ovarian cancer cells need to be fractionated into: mitochondrial and cytoplasmic fractions because MNRR1 can be found/functions in the mitochondria and/or nucleus.
2. BCA: To get protein concentration before running a Western Blot.
3. Western Blot: To find if the MNRR1 is found in the nucleus and/or mitochondria of different ovarian cancer cells

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Method Number 1 (cont.)

• TKO (mouse ovarian cancer cells)
• OVCA432 (human ovarian cancer cells)
• R182 (human ovarian cancer cells)
• F2 (human ovarian cancer cells)

Conclusion 1

• In the TKO cell line majority of the MNRR1 is present in the cytoplasmic/ mitochondrial fraction
• In the F2 cell line we can not conclude whether there is more of the MNRR1 in the cytoplasmic/ mitochondrial or nuclear fraction because of the contamination in the nuclear fraction.

Method Number 2

• What is Immunoprecipitation?
• It is a process used to separate a protein from the rest of the proteins contained in a sample

Method Number 2 (cont.)

• Experimental design (second): Immunoprecipitation (IP)
1. Protein Lysing: To break down the different ovarian cancer cells and capture the proteins for analyzation
2. BCA: To get protein concentration before running a Western Blot
3. Immunoprecipitation: To isolate MNRR1 and what is bound to it in different ovarian cancer cells
4. Western Blot: To detect what MNRR1 is bound to (BCLXL and/or COX4)

Method Number 2 (cont.)

• When MNRR1 is bound to BCLXL it causes anti apoptosis
• When MNRR1 is bound to COX4 it induces cellular respiration

Method Number 2 (cont.)

• TKO (mouse)
• mR182
• F2

F2: IP IGG

F2: IP COXIV

Input

Supt

Wash

IP

Supt

Wash

IP

MNRR1

COXIV

F2: IP IGG

F2: IP BCLXL

Input

Supt

Wash

IP

Supt

Wash

IP

MNRR1

BCLXL

TKO: IP IGG

TKO: IP COXIV

Input

Supt

Wash

IP

Supt

Wash

IP

MNRR1

COXIV

TKO: IP IGG

TKO: IP BCLXL

Input

Supt

Wash

IP

Supt

Wash

IP

MNRR1

BCLXL

Conclusion 2

• In the TKO cell line MNRR1 is shown to be bound to BCLXL
• In the F2 cell line the results are inconclusive

General Conclusion

• In TKO most of the MNRR1 is found in the mitochondria and most of the MNRR1 is shown to be bound to BCLXL
• In F2 MNRR1 is present in both fractions but it is not clear where a majority of it is located. The results could not demonstrate whether it binds to COXIV or BCLXL.