<p>Breast cancer is the
predominant cancer diagnosed among women, and the second most deadly cancer.
The vast majority of cancer-related deaths is caused by the metastatic spread
of cancer from the primary tumor to a distant site in the body. Therefore, new
strategies which minimize breast cancer metastasis are imperative to improve
patient survival. Cancer cells which acquire anchorage independence, or the
ability to survive without extracellular matrix attachment, and metabolic flexibility
have increased potential to metastasize. In the present studies, the ability to
survive detachment and subsequent metabolic changes were determined in human
Harvey-<i>ras</i> transformed MCF10A-<i>ras</i> breast cancer cells. Detachment
resulted in reduced viability in a time-dependent manner with the lowest cell
viability observed at forty hours. In addition, decreased cell viability was
observed in both glutamine and glucose depleted detached conditions, suggesting
a dependence on both nutrients for detached survival. Compared to attached
cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine,
serine, and glutamate, suggesting the metabolic stress which occurs under
detached conditions. However, intracellular citrate and aspartate pools were
unchanged, demonstrating a preference to maintain these pools in detached
conditions. Compared to attached cells, detached cells had suppressed glutamine
metabolism, as determined by decreased glutamine flux into the TCA cycle and
reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached
glucose anaplerosis through pyruvate dehydrogenase activity was decreased,
while pyruvate carboxylase (PC) expression and activity were increased. A
switch in metabolism was observed away from glutamine anaplerosis to a
preferential utilization of PC activity to replenish the TCA cycle, determined
by reduced PC mRNA abundance in detached cells treated with a cell-permeable
analog of α-ketoglutarate,
the downstream metabolite of glutamine which enters the TCA cycle. These
results suggest that detached cells elevate PC to increase flux of carbons into
the TCA cycle when glutamine metabolism is reduced. </p>
<p>Vitamin D is recognized for its role in preventing breast cancer
progression, and recent studies suggest that regulation of energy metabolism
may contribute to its anticancer effects. Vitamin D primarily acts on target
tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)<sub>2</sub>D). The present work
investigated 1,25(OH)<sub>2</sub>D’s effects on viability of detached cells
through regulation of energy metabolism. Treatment of MCF10A-<i>ras</i> cells
with 1,25(OH)<sub>2</sub>D resulted in decreased viability of detached cells.
While 1,25(OH)<sub>2</sub>D treatment did not affect many of the glucose
metabolism outcomes measured, including intracellular pyruvate and lactate pool
sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes
involved in glucose metabolism, 1,25(OH)<sub>2</sub>D treatment reduced detached
PC expression and glucose flux through PC. A reduction in glutamine metabolism
was observed with 1,25(OH)<sub>2</sub>D treatment, although no 1,25(OH)<sub>2</sub>D
target genes were identified. Further, PC depletion by shRNA decreased cell
viability in detached conditions with no additional effect with 1,25(OH)<sub>2</sub>D
treatment. Moreover, PC overexpression resulted in increased detached cell
viability and inhibited 1,25(OH)<sub>2</sub>D’s negative effects on viability.
These results suggest that 1,25(OH)<sub>2</sub>D reduces detached cell
viability through regulation of PC. Collectively this work identifies a key
metabolic adaptation where detached cells increase PC expression and activity
to compensate for reduced glutamine metabolism and that 1,25(OH)<sub>2</sub>D
may be utilized to reverse this effect and decrease detached cell viability.
These results contribute to an increased understanding of metastatic processes
and the regulation of these processes by vitamin D, which may be effective in
preventing metastasis and improve breast cancer patient survival.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14456148 |
| Date | 07 May 2021 |
| Creators | Madeline Sheeley (10676388) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Regulation_of_Energy_Metabolism_in_Extracellular_Matrix_Detached_Breast_Cancer_Cells/14456148 |
Page generated in 0.0024 seconds