CREB3L1 as a potential biomarker predicting response of triple negative breast cancer to doxorubicin-based chemotherapy

Breast cancer can be divided into subgroups based on expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, which are growth factor receptors that are crucial for proliferation of cancer cells. While targeted therapies antagonizing these receptors have significantly improved the treatment of breast cancers expressing these receptors, treating those that express none of the three receptors, namely triple negative breast cancer (TNBC), remains a daunting challenge [1]. Chemotherapy is still the mainstream treatment for these cancers. Nearly all chemotherapy regimens used to treat TNBC contain multiple drugs, and the majority of those include doxorubicin due to its effectiveness demonstrated through decades of clinical practice [2]. However, the response rate of doxorubicin-based chemotherapy is only 40–70%, primarily because there is no biomarker capable of identifying patients who will respond to doxorubicin [3–5]. Considering the severe cardiac toxicity caused by the side effect of doxorubicin [6], establishment of such a biomarker will not only improve the response rate of doxorubicin-based chemotherapy, but also reduce the toxicity of the treatment by avoiding application of doxorubicin to patients who are unlikely to respond to doxorubicin.

Doxorubicin has been demonstrated to induce DNA damage by inhibiting topoisomerase II, but whether this activity is associated with its ability to suppress cancer cell proliferation is still under debate [7, 8]. We have recently reported that doxorubicin blocks proliferation of cancer cells through activation of a transcription factor called cAMP response element binding protein 3-like 1 (CREB3L1) [9]. Unlike most transcription factors, CREB3L1 is synthesized as an inactive transmembrane precursor, with the transcriptionally active N-terminal domain anchored to membranes via a single transmembrane helix [10]. Doxorubicin stimulates proteolytic cleavage of CREB3L1, releasing the N-terminal domain of the protein from membranes, allowing it to enter nucleus where it activates transcription of genes that inhibit cell proliferation [9, 10]. As a result, doxorubicin inhibited proliferation of tumor cells cultured in vitro that express CREB3L1 but not those in which expression of the gene was inhibited, even though DNA damage induced by the drug was indistinguishable among these cells [9]. Using a mouse xenograft model of human renal cell carcinoma reported to maintain drug sensitivities displayed in patients [11], we previously reported that doxorubicin at a dose lower than that typically applied to human patients shrank tumors expressing high levels of CREB3L1 but not those expressing low levels of the protein [12]. Notably, at this low dose doxorubicin did not induce DNA damage in the xenograft tumors regardless of their CREB3L1 expression levels [12]. These in vitro and in vivo results suggest that doxorubicin inhibits tumor cell proliferation through proteolytic activation of CREB3L1 but not DNA damage. These findings demonstrate that CREB3L1 expression in cancer cells could be a key determinant for their sensitivity to doxorubicin.

In the current study, we performed a retrospective analysis on core biopsy samples taken from TNBC patients before chemotherapy to measure CREB3L1 expression levels in cancer cells, and to determine the relationship between CREB3L1 expression levels and outcomes of doxorubicin-based chemotherapy. The results showed that CREB3L1 expression levels in cancers that responded to chemotherapy were significantly higher than those that resisted the treatment. Our findings suggest that high levels of CREB3L1 expression in cancer cells could serve as a predictive biomarker to identify TNBC patients who are likely to respond to doxorubicin-based chemotherapy.

A specific antibody against CREB3L1 without significant cross reactivity is required for IHC analysis of CREB3L1. The monoclonal antibody 10H1 we raised previously [12] is suitable for this application: On immunoblot the antibody detected only the precursor and cleaved form of CREB3L1 (Fig. 1, lane 1) in Huh7 cells stably transfected with a control shRNA that expressed CREB3L1, but detected no protein in Huh7 cells stably transfected with a shRNA targeting CREB3L1 that inhibited expression of the mRNA by more than 90% [9] (Fig. 1, lane 2). In contrast to 10H1, a commercially available anti-CREB3L1 stated suitable for IHC had much more cross reactivity (Fig. 1, lanes 3 and 4).

We then obtained unstained slides of core biopsies taken from TNBC patients before they were subjected to doxorubicin-based chemotherapy, and used 10H1 to measure CREB3L1 expression in the tumor cells through IHC. Some typical results of the IHC analysis were shown in Fig. 2, which include TNBC with no detectable expression (Fig. 2a), with heterogeneous expression (Fig. 2b), or with homogenous high levels of expression of CREB3L1 (Fig. 2c).

We quantified CREB3L1 expression in cancer cells of all patients before doxorubicin-based chemotherapy through the IRS system, and measured their response to the following chemotherapy through the RCB system. In the RCB system, the higher the score, the more resistant the tumor to chemotherapy [14]. Based on the RCB scores, the tumors are divided into 4 classes, with class 0, 1-2, and 3 refers to completely responsive, partially responsive, and completely resistant tumors, respectively [14]. The results were summarized in Table 1. According to our hypothesis, higher levels of CREB3L1 expression should render cancer cells more sensitive to doxorubicin. Consistent with this hypothesis, we observed that the average IRS value of tumors that completely or partially responded to the chemotherapy (RCB classes 0-2) was 8 times higher than that of the tumors resistant to the treatment (RCB class 3) (4.8 vs 0.6) (Fig. 3a). This difference is statistically significant indicated by the student t-test (p = 0.0005) and power analysis (statistical power 99.8% assuming a 95% confidence interval). Likewise, the degree of resistance to the chemotherapy measured by increased RCB scores was negatively correlated with CREB3L1 expression levels (Pearson analysis, R = -0.54, p = 0.02) (Fig. 3b).

Case	Age	Chemotherapy in Addition to Doxorubicin	Pathologic TNM	Stage	Ki67	RCB Score	RCB Class	CREB3L1 IRS
1	40-49	Cytoxan, Taxol, Xeloda	ypT2pN3M1	IV	84	4.186	3	1
2	50-59	Cytoxan, Xeloda, Taxol	ypT2pN1M1	IV	45	4.387	3	0.5
3	50-59	Cytoxan, Taxol	ypT1bN0M0	IA	90	1.340	1	0.5
4	30-39	Cytoxan, Taxol	ypT2N3M0	IIIC	70	4.340	3	1
5	40-49	Cytoxan, Taxol	ypT0N1miM0	IB	95	1.125	1	10
6	50-59	Halaven, Paraplatin, Taxol	ypT2N1M1	IV	90	3.290	3	0.5
7	50-59	Cytoxan, Paraplatin, Taxol	ypT1cN1M0	IIA	30	3.014	2	6
8	40-49	Cytoxan, Taxol	ypT0N0M0	0	95	0	0	8
9	40-49	Cytoxan, Paraplatin, Taxol, Xeloda	ypT2N1M1	IV	35	1.390	2	2
10	40-49	Cytoxan, Gemzar, Paraplatin, Taxol	ypT1bN1M0	IIA	20	3.023	2	8
11	40-49	Cytoxan, Taxol	ypT2N0M0	0	60	0	0	2
12a	50-59	Arimidex, Cytoxan, Paraplatin, Taxol	ypT2N1M0	IIB	80	3.900	3	0
13	40-49	Cytoxan, Taxol, Xeloda	ypT2N0M0	0	85	2.452	2	1
14	70-79	Cytoxan, Taxol	ypT0N0M0	0	90	0	0	6
15	50-59	Cytoxan, Taxol	ypT1miN0M0	0	95	1.322	1	6
16a	50-59	Arimidex, Cytoxan, Taxol	ypT1cN0M0	IA	95	2.009	2	4
17a	40-49	Arimidex, Cytoxan, Taxol	ypT1cN0M0	IA	70	2.140	2	1
18a	60-69	Arimidex, Cytoxan, Taxol	ypTisN0M0	0	80	0	0	8

All patients were diagnosed and treated between 2011 and 2017 at UT Southwestern University Hospitals. Patient age range and chemotherapeutic drugs in addition to doxorubicin used in the treatment are shown. Response of the tumors to the chemotherapy measured through RCB Scores and RCB Classes, CREB3L1 expression in the tumors measured by IRS values, percentage of tumor cells positively stained by anti-Ki67, and other clinical characterization of the tumors is also presented

^aThese patients had two different breast cancers: one breast with TNBC; the other with ER positive cancer. Only TNBC but not the ER-positive tumors were analyzed in this study

To determine whether CREB3L1 expression levels can serve as a biomarker to predict outcomes of doxorubicin-based chemotherapy, we separated the tumors into two groups: One with lower (IRS ranges from 0 to 3) and the other with higher (IRS ranges from 4 to 12) levels of CREB3L1 expression. We observed that all tumors resistant to doxorubicin-based chemotherapy fell in the group with lower expression of CREB3L1 (Fig. 3c). As a result, only 50% of the tumors in this group responded to the chemotherapy (Fig. 3c). In contrast, all tumors with higher expression of CREB3L1 were responsive to the chemotherapy (Fig. 3c).

We have previously reported that doxorubicin inhibits proliferation of tumor cells through proteolytic activation of CREB3L1, and CREB3L1 expression determines the sensitivity of tumors to doxorubicin in various in vitro and in vivo experimental systems [9, 12]. The current study demonstrates the clinical importance of the finding by establishing CREB3L1 expression as a potential biomarker capable of identifying TNBC patients who are likely to benefit from doxorubicin-based chemotherapy. We show that tumors with higher levels of CREB3L1 expression (IRS value ≥ 4) all responded to doxorubicin-based chemotherapy. Thus, applying such chemotherapy only to patients with higher levels of CREB3L1 expression should markedly increase the response rate of the treatment. We also observe that all tumors resistant to doxorubicin-based chemotherapy had lower levels of CREB3L1 expression (IRS value ≤ 3). However, some tumors with lower levels of CREB3L1 expression did respond to the chemotherapy. The most likely explanation for this observation is that TNBC patients are not treated with doxorubicin alone, and these tumors may be sensitive to other drugs included in the chemotherapy regimens such as Cytoxan and Taxol. If this is the case, then for tumors with lower levels of CREB3L1 expression, replacing doxorubicin with other chemotherapeutic reagents could prevent unnecessary cardiac toxicity associated with doxorubicin [6] without affecting the response rate of the treatment.

The current study, in which only 18 samples were analyzed, was originally designed as a pilot study to estimate the sample size required to reach statistical significance. Surprisingly, our small sample number was sufficient for us to draw the conclusion that CREB3L1 expression levels in tumors responsive to doxorubicin-based chemotherapy were higher than those resistant to the treatment. This is because the difference in CREB3L1 expression levels between the two groups of the tumors was so large (~ 8 fold) that even with such a limited sample size, statistical significance was achieved (p = 0.0005) though a simple student t-test. More importantly, power analysis, which has been established as the standard for sample size estimation in clinical studies [16], demonstrated that our statistic power (99.8%) was much higher than the value (80%) considered statistically significant in most clinical studies. Despite the small sample size, our sample selection was not skewed in terms of response of the tumors to chemotherapy, as the percentage of cases completely responsive or resistant to the therapy in our samples was similar to that in clinical trials involving hundreds of patients [3, 17]. Thus, adding more samples to this retrospect study will not further improve statistical significance in a clinically meaningful manner. Instead, this study should serve as a base for future prospective studies to firmly establish CREB3L1 as a clinical biomarker to identify TNBC patients who are most likely to respond to doxorubicin-based chemotherapy.

The current study reveals that CREB3L1 expression varies widely among different TNBC tumors. About 50% of the tumors we examined had low levels of CREB3L1 expression (IRS value ≤ 3). Previous reports have indicated that expression of CREB3L1 is frequently epigenetically silenced through DNA methylation in various cancers including breast cancer [18–21]. We have previously reported that azacitidine, a drug that inhibits DNA methylation [22], relieved epigenetic silencing of CREB3L1 [18]. In cultured MCF-7 cells in which CREB3L1 expression is barely detectable [9], azacitidine markedly increase the sensitivity of the cells to doxorubicin [23]. It will be interesting to determine whether addition of azacitidine to doxorubicin-based chemotherapy regimens may improve the treatment outcomes for TNBC with lower levels of CREB3L1 expression.

The current study demonstrates that TNBC tumors with higher expression of CREB3L1 have a higher response rate to doxorubicin-based chemotherapy compared to those with lower expression of the protein. These results suggest that CREB3L1 expression level may serve as a biomarker to identify TNBC patients who are more likely to benefit from doxorubicin-based chemotherapy.