Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma
Yulong Chen, … , Mitsuo Yamauchi, Jonathan M. Kurie
Yulong Chen, … , Mitsuo Yamauchi, Jonathan M. Kurie
Published February 9, 2015
Citation Information: J Clin Invest. 2015;125(3):1147-1162. https://doi.org/10.1172/JCI74725.
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Research Article Oncology

Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma

Abstract

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde–derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

Authors

Yulong Chen, Masahiko Terajima, Yanan Yang, Li Sun, Young-Ho Ahn, Daniela Pankova, Daniel S. Puperi, Takeshi Watanabe, Min P. Kim, Shanda H. Blackmon, Jaime Rodriguez, Hui Liu, Carmen Behrens, Ignacio I. Wistuba, Rosalba Minelli, Kenneth L. Scott, Johannah Sanchez-Adams, Farshid Guilak, Debananda Pati, Nishan Thilaganathan, Alan R. Burns, Chad J. Creighton, Elisabeth D. Martinez, Tomasz Zal, K. Jane Grande-Allen, Mitsuo Yamauchi, Jonathan M. Kurie

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Figure 6

STAT3-induced LH2 expression is HIF-1 dependent.

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STAT3-induced LH2 expression is HIF-1 dependent. (A) LH2 gene promoter f...
(A) LH2 gene promoter fragments created by PCR contain the predicted transcription factor binding sites (color-coded) (JASPAR: http://jaspar.genereg.net/ and BIOBASE: http://www.biobase-international.com/). A549 cells transiently cotransfected with an empty (vec) or constitutively active (CA) mutant STAT3 expression vector, a Renilla luciferase reporter (for normalization), and a firefly luciferase reporter driven by LH2 promoter constructs. Firefly luciferase activities from a single experiment (bar graphs aligned with promoter constructs) expressed as mean ± SD of triplicate wells relative to the activity of the basal reporter (pGL3), which was set at 1.0. (B) HIF-1α levels determined by ELISA on A549 cell lysates stably transfected with HIF-1α or control (FF2) shRNA. Data from a single experiment are expressed as mean ± SD of triplicate samples. (C) Q-PCR analysis of LH2 expression in A549 cells stably transfected with HIF-1α shRNA (SHA or SHE) or control shRNA (FF2). Normalized Q-PCR results from a single experiment are expressed as mean ± SD of triplicate samples. (D) ChIP assay performed on A549 cell lysates using anti–HIF-1α and -STAT3 antibodies. Precipitated DNA amounts (percent of input) expressed as mean ± SD of triplicate samples from a single experiment. IgG was used as a negative control. (E) Luciferase assays performed in a single experiment on A549 cells as described in A. LH2 promoter reporter constructs contain site-directed mutations in HIF-1α (HIF-1-m) or Egr1 (Egr1-m) binding sites or deletion of both sites (DEL). P values, 2-tailed Student’s t test.