SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization
Tatsuya Suwa, … , Ester M. Hammond, Hiroshi Harada
Tatsuya Suwa, … , Ester M. Hammond, Hiroshi Harada
Published November 8, 2021
Citation Information: JCI Insight. 2021;6(21):e148135. https://doi.org/10.1172/jci.insight.148135.
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Research Article Cell biology Oncology

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

Abstract

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2–related factor 2–dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia.

Authors

Tatsuya Suwa, Minoru Kobayashi, Yukari Shirai, Jin-Min Nam, Yoshiaki Tabuchi, Norihiko Takeda, Shusuke Akamatsu, Osamu Ogawa, Takashi Mizowaki, Ester M. Hammond, Hiroshi Harada

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Figure 2

Transcription of the SPINK1 gene is upregulated under hypoxia in a HIF-dependent manner.

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Transcription of the SPINK1 gene is upregulated under hypoxia in a HIF-d...
(A–D) HeLa cells were cultured under the indicated oxygen conditions in the presence or absence of 5 μg/mL Act D for 24 hours (A), 100 μM deferoxamine for 48 hours (B), 2 mM DMOG for 24 hours (C), or 3 μM MG132 for 12 hours (D) and subjected to qPCR. (E–G) HeLa cells were transfected with the indicated siRNA or scramble siRNA for negative control, cultured under the indicated oxygen conditions for 24 hours, and subjected to qPCR. (H and I) After simultaneously silencing HIF-1α, HIF-2α, and HIF-3α using 2 kinds of mixtures using HIF-αs and siRNAs (mixture-1 and mixture-2), HeLa cells were cultured under the indicated oxygen conditions for 24 hours and subjected to qPCR (H) or the ELISA assay (I). (J and K) The same experiments as in H and I were conducted after silencing HIF-1β. Scramble siRNA was used as a negative control. Data are represented as mean ± SD (n = 3). Two-tailed Student’s t test (A–D). One-way ANOVA with Dunnett’s test (E–K). *P < 0.05, **P < 0.01, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; Act D, actinomycin D; DMOG, dimethyloxallyl glycine.