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Role of malignant microcalcifications in HER2+ breast cancer cells

Zhang, He (2022) Role of malignant microcalcifications in HER2+ breast cancer cells. Master's Thesis, University of Pittsburgh. (Unpublished)

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Breast microcalcifications (MCs) are tiny calcium deposits that appear on mammograms and are commonly used as a diagnostic tool for nonpalpable early-stage breast cancer. Based on the chemical composition, MCs are categorized into type I calcium oxalate (OX, benign); and type II calcium phosphate, mainly hydroxyapatite (HA, malignant) microcalcifications. Clinical studies show that calcium phosphate MCs are more likely to be present in human epidermal growth factor receptor 2 (HER2) positive breast cancer. Patient biopsies also show that the breast cancer cells surrounding these MCs express higher levels of mesenchymal markers and bone markers, suggesting the possibility of epithelial to mesenchymal transition (EMT) and bone mimicry.

Abnormal inorganic phosphate serum concentration caused by hyperphosphatemia or high phosphate diet is associated to calcifications in multiple soft tissues. Interestingly, studies unrelated to MCs have shown higher phosphate concentration in breast tumor microenvironment. Growth rate hypothesis also indicates higher local phosphate concentration in tumor microenvironment to sustain high proliferation rate of tumor cells. Thus, it is plausible that the presence of malignant calcium phosphate MCs creates phosphate-rich microenvironment in tumor.

Inorganic phosphate concentration is regulated by sodium-dependent phosphate (Na-Pi) cotransporters, namely SLC34A2, SLC20A1 (Pit1) and SLCA0A2 (Pit2). In particular, SLC34A2 regulates the phosphate concentration in multiple lumen fluids including mammary glands and maintains phosphate homeostasis, whereas the SLC20 family regulates cellular phosphate concentration with a potential role in phosphate sensing. Elevated levels of Pi and Na-Pi co-transporters are known to play a critical role during bone development and bone diseases. Several studies have shown abnormal expression of Na-Pi cotransporters in multiple tumor types and pathological calcification. However, not much is known about the effect of malignant calcium phosphate MCs on regulating the expression of Na-Pi cotransporter expression in breast cancer cells.

In this study, we hypothesized that the malignant calcium phosphate MCs can create a phosphate rich microenvironment, which may alter Pi transport and expression of Na-Pi cotransporters in breast cancer cells and induce EMT process and bone mimicry in the surrounding breast cancer cells. To test our hypothesis, we selected two breast cancer cell lines with different ER, HER2 status: BT474 (ER+, HER2+) and SUM225 (ER-, HER2+), and then compared expression of Na-Pi co-transporters with normal human breast cancer epithelial cells MCF10A (ER-, HER2-). Then, we fabricated extracellular matrix (ECM)-mimicking soft hydrogels (ECM-mimics) to deposit and recapitulate chemical composition of benign (OX) and malignant (HA) MCs found in breast cancer patients.

Our results showed the mRNA expression of Na-Pi co-transporters was altered depending on the ER, HER2 status of breast cancer cells. Pre-existing malignant (calcium phosphate) MCs did not have significant effect on ER, HER2 status in both cell lines. when exposed to the pre-existing malignant (calcium phosphate) MCs in the ECM-mimics, ER+, HER2+ BT474 cells exhibit reduced expression of epithelial marker, E-cadherin and increased expression of bone marker, BMP2, while upregulating the expression of Pit1 and SLC34A2 compared to BT474 cells seeded on non-mineralized or benign MC-containing ECM-mimics. On the other hand, ER-, HER2+ SUM225 cells seeded on the pre-existing malignant (calcium phosphate) MC-containing ECM-mimics exhibit increased expression of vimentin and BMP2, with no significant change on the expression of Na-Pi co-transporters compared to SUM225 cells seeded on non-mineralized or benign MC-containing ECM-mimics.

In conclusion, this study indicates that presence of malignant MCs may alter expression of Na-Pi co-transporters, and this may be dependent on the ER or HER2 status of the cancer cells.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Zhang, Hehez18@pitt.eduhez18
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSant, Shilpashs149@pitt.edushs149
Committee MemberLi, Songsol4@pitt.edusol4
Committee MemberRohan, Lisa Cenciarohanlc@upmc.edurohanl
Date: 22 April 2022
Date Type: Publication
Defense Date: 28 March 2022
Approval Date: 22 April 2022
Submission Date: 20 April 2022
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 69
Institution: University of Pittsburgh
Schools and Programs: School of Pharmacy > Pharmaceutical Sciences
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Breast cancer; Na-Pi co-transporters; Microcalcifications; Epithelial to mesenchymal transition; Bone mimicry; Human epidermal growth factor receptor 2.
Date Deposited: 22 Apr 2022 17:07
Last Modified: 22 Apr 2022 17:07


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