Noah PrinceAuthentic Science Research Program

Manchester-Essex Regional High School, Manchester-by-the-Sea, MA 01944

Influence of hypoxic conditions on expression of genes related to metabolism in cancer cells

Fig 4 & 5. I used a Roche LightCycler 480 Real-Time PCR System with a SYBR Green 1 Master Mix and protocol for my qPCR assays (Fig 4). For these assays, I would use a 96 well plate (Fig 5).

Materials and Methods• Cell Culture

• Cultured cell lines at 2 different oxygen levels • 21% oxygen – normal atmospheric conditions (normoxia)• 1% oxygen – normal conditions for a growing tumor (hypoxia)

•Cell Lines• SUM159 - Human breast cancer line• flopcRAS

• Engineered cancer cell line• Cells with RAS gene spliced in

• RNA Harvesting• cDNA Synthesis• qPCR to determine levels of gene expression

Fig 3. Image of SUM159 cells

My Project

IntroductionIt is a well known fact that cancer patients experience massive weight loss. This is thought to be a side effect of both treatment and the cancer itself. It is possible, however, that weight loss could be caused directly by tumors. Specifically, tumors could release factors that order the host body to release adipose fat from storage for use by the tumor.

Figures 1 & 2. Fig 1 on the left depicts a section of a bone in a healthy mouse; white spots show ample adipose cells. Fig 2 on the right depicts a section of a bone from a mouse with a distal tumor. Note the lack of adipocytes in the mouse with the cancer.

PurposeTo determine whether oxygen levels could affect the expression of specific genes of interest (GOI). All GOI were proteins related to lipid transport.

Results• There was a difference in levels of gene expression

between hypoxia and normoxia cells• In the sum159 cells, FABP3 and LPL were consistently

upregulated• In both cell lines, SLC27A2 was consistently down-

regulated159-ACTB

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SUM159 NormoxicSUM159 Hypoxic

159-RPL27

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SUM159 NormoxicSUM159 Hypoxic

159-HPRT1

0

5

10

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SUM159 NormoxicSUM159 Hypoxic

159-GUSB

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5

10

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SUM159 NormoxicSUM159 Hypoxic

159-PPIA

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10

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SUM159 NormoxicSUM159 Hypoxic

flopcRAS-ACTNB

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5

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flopcRAS NormoxicflopcRAS Hypoxic

flopcRAS-RPL27

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flopcRAS NormoxicflopcRAS Hypoxic

flopcRAS-HPRT1

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5

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flopcRAS NormoxicflopcRAS Hypoxic

flopcRAS-GUSB

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5

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flopcRAS NormoxicflopcRAS Hypoxic

flopcRAS-PPIA

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5

10

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flopcRAS NormoxicflopcRAS Hypoxic

• In the flopcRAS cells, VLDLR 1 and 2 FABP3, and FABP5 were consistently up-regulated

• In both cell lines, SLC27A2 was consistently down-regulated

Conclusions• Significant differences in expression of GOIs

between the hypoxia and normoxia cells

• Subsequent experiments need to be done with hypoxic cells to better simulate the tumor environment

• Hypoxic conditions stimulate cancer cells to be more dependant upon lipids.

• Hypoxia plays a role in how cancer cells work.

Literature citedBennani-Baiti N, Walsh D. What is cancer anorexia-

cachexia syndrome? A historical perspective. J R Coll Physicians Edinb. 2009 Sep;39(3):257-62.

Grossberg AJ, Scarlett JM, Marks DL. Hypothalamic mechanisms in cachexia, Physiol Behav. 2010 Jul 14;100(5):478-89. Epub 2010 Mar 25.

Nomura DK, Long JZ, Niessen S, Hoover HS, Ng S, Cravatt BF. Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis, Cell 140, 49–61, January 8, 2010

AcknowledgmentsI thank Jordan Krall for being a great mentor, Robert Weinberg for giving me my internship, and Maria Burgess for helping me every step of the way. Supported by a grant from the Spaulding Education Fund.

AbstractInfluence of Hypoxic Conditions on Expression of Genes Related to Metabolism in Cancer Cells Noah PrinceManchester-Essex High School, Manchester-by-the-Sea, MATeacher, Dr. Maria Burgess, Manchester-Essex High SchoolMentor, Dr. Jordan Krall, Whitehead Institute, Cambridge, MA

Although it is thought to be a side effect of cancer, large amounts of weight loss might be a direct attack on the body by malignant tumors. It might be possible that weight, and specifically adipocyte loss, might be caused directly by the tumor itself. Stresses, such as hypoxic conditions, on cancers can cause large changes in gene expression, and it is possible that hypoxic conditions are what cause the tumor’s increased appetite for adipocytes. It was found that there were definite changes in the expression of genes related to metabolism, such as FABP3, FABP5, LPL, and VLDLRV2, between cells exposed to hypoxic conditions and cells exposed to normoxic conditions. This data shows that differing levels of oxygen do have an affect on cancer cells’ dependency on lipids.