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The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth
Lilly He1, Keiko Takahashi1, Lejla Pasic2
1Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Background:
CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied.
Aim:
We aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling.
Methods:
CD148 forms, wild-type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin-1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme-linked immunosorbent assay, and Western blotting.
Results:
Both CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF-induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)-expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells.
Conclusion:
Our data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1-CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.
Insights
The CD148 protein tyrosine phosphatase mutations Q276P/R326Q do not significantly alter its ability to inhibit cancer cell proliferation or epidermal growth factor receptor (EGFR) signaling. These findings suggest the mutations do not impact CD148
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- CD148 is a transmembrane protein tyrosine phosphatase regulating cell growth by dephosphorylating growth factor receptors.
- CD148 gene polymorphisms (Q276P, R326Q) are linked to increased cancer risk.
- The biological impact of these specific CD148 mutations remains unstudied.
Purpose of the Study:
- To investigate the biological effects of CD148 Q276P/R326Q mutations.
- To assess the impact on cancer cell proliferation and epidermal growth factor receptor (EGFR) signaling.
- To understand the role of these mutations in CD148's interaction with its ligand, thrombospondin-1 (TSP1).
Main Methods:
- Engineered A431D epidermoid carcinoma cells lacking CD148 expression with wild-type (WT) or Q276P/R326Q CD148 forms.
- Utilized flow cytometry for cell sorting and comparable expression levels.
- Assessed CD148 localization, cell proliferation, EGFR signaling, and TSP1 response via immunostaining, proliferation assays, ELISA, and Western blotting.
Main Results:
- Both WT and mutant CD148 localized to the cell surface and reduced cancer cell proliferation.
- EGF-induced EGFR and ERK1/2 phosphorylation were significantly reduced in cells expressing CD148.
- TSP1 inhibited proliferation in CD148-expressing cells, but no significant differences were found between WT and mutant CD148 cells.
Conclusions:
- CD148 Q276P/R326Q mutations do not substantially affect CD148's interaction with TSP1.
- The mutations do not alter CD148's cellular localization or its inhibitory activity on EGFR signaling and cell proliferation.
- The studied CD148 mutations do not appear to be the primary drivers of the observed increased cancer risk associated with these polymorphisms.
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