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 Article has an altmetric score of 6

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 1

SPINK1 is identified as a candidate plasma marker for tumor hypoxia and potential therapeutic target for radiosensitization.

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SPINK1 is identified as a candidate plasma marker for tumor hypoxia and ...
(A) HeLa cells were cultured under the indicated oxygen conditions for 24 hours and subjected to DNA microarray analysis. Of 34 genes that exhibited more than 10-fold induction upon hypoxia, the top 4 genes harboring the N-terminus signal peptide are listed. (B and C) HeLa cells were cultured under the indicated oxygen conditions for the indicated periods, and subjected to qPCR for the indicated genes. (D) After being cultured under the indicated oxygen conditions for 48 hours, cell lysates were subjected to qPCR. (E) Changes in the SPINK1 mRNA levels in HeLa cells were quantified at the indicated time points during (prehypoxia) and after (reoxygenation) the severe hypoxic treatment and represented as mean ± SD. (F) After the same treatment as in D, culture media were subjected to the ELISA assay. (G) Scatter plot for correlation analysis between SPINK1 mRNA levels and secreted SPINK1 protein levels in cells cultured under the indicated oxygen conditions for the indicated periods. (H–K) The indicated cells were transfected with either pcDNA4/SPINK1 (SPINK1) or its EV and cultured for 48 hours. Then, both culture media and cell lysates were subjected to Western blotting using the indicated antibodies (H and J), and then, cells were irradiated with the indicated doses of γ-rays and subjected to the clonogenic survival assay (I and K). The exogenously expressed SPINK1 was detected using anti-myc tag Ab (H and J). Data are represented as mean ± SD (B, D, F, I, and K; n = 3 in B–G, n = 6 in I and K). Two-tailed Student’s t test. *P < 0.05, ***P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.