Identification of CREB3L1 as a Biomarker Predicting Doxorubicin Treatment Outcome.

Doxorubicin has been shown to inhibit proliferation of cancer cells through proteolytic activation of CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor. Upon doxorubicin treatment, CREB3L1 is cleaved so that the N-terminal domain of the protein can reach the nucleus where it activates transcription of genes that inhibit cell proliferation. These results suggest that the level of CREB3L1 in cancer cells may determine their sensitivity to doxorubicin.Mice transplanted with 6 lines of renal cell carcinoma (RCC) were injected with doxorubicin to observe the effect of the chemotherapy on tumor growth. Immunohistochemistry and bioinformatics analyses were performed to compare CREB3L1 levels in types of cancer known to respond to doxorubicin versus those resistant to doxorubicin.Higher levels of CREB3L1 protein are correlated with increased doxorubicin sensitivity of xenograft RCC tumors (p = 0.017 by Pearson analysis). From patient tumor biopsies we analyzed, CREB3L1 was expressed in 19% of RCC, which is generally resistant to doxorubicin, but in 70% of diffuse large B-cell lymphoma that is sensitive to doxorubicin. Doxorubicin is used as the standard treatment for cancers that express the highest levels of CREB3L1 such as osteosarcoma and malignant fibrous histiocytoma but is not generally used to treat those that express the lowest levels of CREB3L1 such as RCC.Identification of CREB3L1 as the biomarker for doxorubicin sensitivity may markedly improve the doxorubicin response rate by applying doxorubicin only to patients with cancers expressing CREB3L1

Clinical application of doxorubicin to cancers with the highest or lowest levels of CREB3L1 expression.

<p>Cancers with the highest or lowest levels of CREB3L1 mRNA were identified through bioinformatics analysis of the Oncomine database. To search for these cancers, p-values were set to less than 10⁻⁴ to minimize variation in CREB3L1 expression among individual tumors in a given type of cancer, and fold of changes were set to more than 3 to select tumors in which levels of CREB3L1 mRNA is at least 3 times higher or lower than the average value of the mRNA across all the tumors deposited into the database. Tumors were ordered by Oncomine according to combined evaluation of fold of changes and p-values. American Cancer Society (<a href="http://www.cancer.org" target="_blank">www.cancer.org</a>) and National Cancer Institute (<a href="http://www.cancer.gov" target="_blank">www.cancer.gov</a>) were consulted to obtain information regarding clinical application of doxorubicin to each cancer.</p><p>^A Doxorubicin is used as a non-standard treatment for ovarian cancers in general, but its effect on ovarian mucinous adenocarcinoma has not been determined.</p><p>Clinical application of doxorubicin to cancers with the highest or lowest levels of CREB3L1 expression.</p

Doxorubicin inhibits growth of tumors expressing high levels of CREB3L1 by stimulating cleavage of CREB3L1.

<p>(A and B) Whole cell lysate (A) or indicated fractions (B) of the RCC xenograft tumors harvested 3 days after the initial injection (2 mice for each tumor) were subjected to immunoblot analysis with the indicated antibodies. Calnexin, lysine-specific demethylase 1 (LSD1) and actin served as loading controls. (C-L) Tumors harvested 21 days after the initial injection were subjected to IHC with anti-endomucin. Representative blood vessels marked by endomucin staining are indicated by arrows. Scale bar: 100 μm. (M) Quantification of blood vessel density in each line of the tumors determined by IHC of endomucin. For each individual tumor, five independent images were taken for calculation. For each line of tumors, results are reported as mean ± S.E. from n ≥ 4 tumors taken from different mice.</p

Response of xenograft tumors formed by Huh7-derived cells to doxorubicin in mice.

<p>(A) CREB3L1 mRNA in Huh7 cells stably transfected with the indicated shRNA was quantified through RT-QPCR with the value in cells transfected with the control shRNA set to 1. Results are reported as mean ± S.E. from three independent experiments. (B) Volume of the xenograft tumors formed by Huh7 cells transfected with the indicated shRNA was measured in mice 3 days after the injection of doxorubicin at 0.75 mg/kg, with the value immediately before the injection set at 100%. Unpaired two-tailed student’s t-test was performed to determine the statistical significance. Similar results were observed in two other independent experiments.</p

Comparison of doxorubicin pharmacokinetic parameters in mice and human patients.

<p>Mice were injected with a single dose of doxorubicin at 5 mg/kg intraperitoneally (IP), and plasma levels of doxorubicin were determined by LC-MS/MS as described in Materials and Methods. The data were fit to a 2-compartment model with bolus input (WinNonlin Model #7) using Phoenix WinNonlin 6.3 software. These data were used to simulate pharmacokinetic parameters for mice injected with doxorubicin at 0.75 mg/kg for 8 times, with the assumption that exposure in mice would be linear in this dose range. Published pharmacokinetic parameters (fit to a 3-compartment model) of human patients injected intravenously (IV) with doxorubicin at a bolus dose of 75 mg/m² is presented for comparison.</p><p>Comparison of doxorubicin pharmacokinetic parameters in mice and human patients.</p

Responses of RCC xenograft tumors to doxorubicin in mice.

<p>(A-F) Volume of the xenograft tumors was measured in mice injected with doxorubicin (0.75 mg/kg), rapamycin (0.5 mg/kg) or the vehicle control once every two days. Duration of each treatment is described in detail in the Method section. The tumor volume immediately before the initial injection is set at 100%. (G) At the end of the experiments 21 days after the initial injection, the effect of doxorubicin on tumor growth expressed as the volume of doxorubicin-treated tumors as a percentage of that treated with the vehicle control was plotted against the relative intensity of CREB3L1 IHC staining in each line of the tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129233#sec008" target="_blank">Methods</a> section for quantification of IHC images). Statistics was performed by Pearson analysis. (H) Weight of the mice transplanted with the indicated tumors and subjected to the indicated treatment was measured at the end of the experiments, with the value immediately before the treatment set at 100%. (A-F, H) Results are reported as mean ± S.E. from n ≥ 4 mice injected with vehicle control or doxorubicin, and n ≥ 3 mice injected with rapamycin. Unpaired two-tailed student’s t-test was performed to determine the statistical significance. *p < 10⁻⁴; **p < 10⁻³; NS, the difference is not statistically significant. Similar results were obtained for XP121, XP164 and XP416 in one other independent experiment.</p

CREB3L1 expression in RCC and DLBCL.

<p>(A-D) Representative IHC images of sections of the indicated tumors stained negatively or positively with anti-CREB3L1. Scale bar: 100 μm. (E) Percentage of indicated tumors stained positively with anti-CREB3L1.</p