Pieter Cory, BS
Pulmonary and Clinical Care Medicine
Pieter Cory*, Dean M. Rosenthal, Nicola Alesi, Damir Khabibullin, Jennifer A. Chen, Michel Alchoueiry, Heng-Jia Liu, Yan Tang, Eli Akl, Julia F. Charles, Volkhard Lindner, Elizabeth P. Henske
Elizabeth P. Henske
Lymphangioleiomyomatosis (LAM) is a destructive cystic lung disease, primarily affecting women of childbearing age. LAM is caused by mutational inactivation of the Tuberous Sclerosis Complex 2 (TSC2) tumor suppressor gene. The mechanisms through which TSC2 mutations result in lung destruction are poorly understood.
Collagen triple helix repeat containing 1 (CTHRC1) is a secreted protein that is highly expressed in many cancers and associated with poor prognosis. Interestingly, CTHRC1 is a hallmark of pulmonary fibrosis-associated fibroblasts, in mice and humans.
We have found that Cthrc1 mRNA expression is elevated in models of LAM and TSC. Cthrc1 mRNA is elevated 15-60-fold in TSC1-/- MEFs, TSC2-/- MEFs, and TSC2-deficient LAM patient-derived 621-102 cells, relative to controls. CTHRC1 protein is also strongly expressed in TSC-deficient cells. Cthrc1 knockdown in Tsc2-/- MEFs decreased proliferation (~2.5-fold, P < 0.0001) and colony formation (~7-fold, P < 0.0001) with no effect on Tsc2+/+ MEFs, indicating that that Cthrc1 drives cell growth in LAM and TSC.
The transcription factors TFE3 and TFEB are hyperactive in TSC and LAM. Cthrc1 contains multiple TFE3/TFEB “CLEAR” motifs upstream of its transcription start site. Consistent with this, Cthrc1 mRNA expression is decreased by TFE3/TFEB knockdown in TSC1-/- MEFs (~2-fold, p < 0.0001) and 621-102 cells (~2.5-fold, p < 0.01).
RNA sequencing revealed a Cthrc1-dependent upregulation of C3 (complement component 3). C3 is a secreted protein vital to innate immunity. RT-qPCR confirmed that C3 mRNA is increased in TSC2-deficient MEFs (~60-fold, p=.003), TSC1-deficient MEFs (3.5-fold, p<.0001), and TSC2-deficient 621-102s (~3.5-fold, P<0.0001) compared to controls.
In aggregate, our results indicate that CTHRC1 expression in TSC/LAM is driven by TFEB/TFE3, and that CTHRC1 promotes cell proliferation and invasion, possibly via upregulation of C3. CTHRC1 expression is insensitive to mTORC1 inhibition, suggesting a novel and key role for CTHRC1 in the pathogenesis and therapy of LAM.
Our results indicate that CTHRC1 expression is driven by TFE3/TFEB activity and may promote proliferation of TSC-deficient cells. CTHRC1’s insensitivity to mTORC1 inhibition via rapamycin suggests a novel and key role for CTHRC1 in the pathogenesis and therapy of LAM/TSC. Because CTHRC1 expression is not affected by rapamycin, the protein could help explain why tumors in LAM/TSC are not eliminated by therapy with mTORC1 inhibitors. Furthermore, our preliminary data on CTHRC1-dependent upregulation of C3 in our TSC-deficient cell models suggests the protein may promote cell proliferation in LAM. Thus, inhibition of C3 may also provide a novel therapy for LAM.