Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer

Abstract

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

Figure 1. Meta-analysis displaying the association of SPINK1 expression with poor DMFS across multiple cohorts (p < 1E−05)

Ten publically available datasets were analysed and are listed on the Y-axis of the graph. The dotted line represents hazard ratio of 1.

Figure 2. Kaplan–Meier curves demonstrating the association of SPINK1 expression with patient prognosis in ER+ and ER− cohorts

Samples are divided into two groups, above mean SPINK1 expression (red) and below mean SPINK1 expression (green). Prognosis is measured by disease and metastasis free survival (years).

1. SPINK1 expression in all samples.

2. ER-negative cases.

3. ER-positive cases.

Figure 3. SPINK1 expression in normal breast and breast tumours

1. SPINK1 expression was negligible in 10 normal breast cores (panels a–h).

2. This panel shows representative cores from the commercial breast TMA. As shown, invasive ductal carcinomas were positive but displayed various levels of SPINK1 (panels a–h).

3. SPINK1 nuclear staining was largely restricted to the breast tumour cells (green arrow) as compared to adjacent normal cells (red arrow).

4. Localization of SPINK1 in vitro. MDA-MB-231 cells were treated with SPINK1-CM and uptake of SPINK1 (if any) was studied using two antibodies. SPINK1 staining observed in cells after 12–24 h of SPINK1 treatment, recapitulating staining observed in primary tumours (Blue: DAPI; Green: anti-SPINK1 antibody; Red: anti-His antibody).

Figure 4. Kaplan–Meier curves demonstrating the association of SPINK1 protein levels with patient prognosis in ER+ and ER− cohorts

Prognosis is measured with DMFS (years).

1. SPINK1 protein expression in all samples.

2. ER-negative cases.

3. ER-positive cases.

Figure 5. Phenotypic assays upon siRNA-knockdown of SPINK1

1. siRNA-mediated knockdown (kd) of SPINK1 using three different siRNAs in breast cancer cell lines MCF-7 and MDA-MB-231. SPINK1 expression was detected via RT-PCR.

2. Effect of SPINK1-kd on cell proliferation in MDA-MB-231 (left panel) and MCF-7 (right panel).

3. Activated PARP and caspase-3 upon siRNA-mediated knockdown of SPINK1 in MDA-MB-231 (left panel) and MCF-7 (right panel). (*p < 0.005; **p < 0.05; ***p < 1E−05).

Figure 6. Proliferation rescue assay of SPINK1 in MDA-MB-231

Incorporation of BrDU in MDA-MB-231 cells upon SPINK1-kd, and rescue with bioactive SPINK1-CM. Significance of knockdown was measured against scrambled control, while significance of SPINK1CM was measured against VecCM as a control (*p < 0.005).

Figure 7. Induction of resistance to chemically induced apoptosis

Activation of PARP (left panel) and caspase-3 (right panel) was measured in MCF-7 vector control (white bars) and SPINK1 overexpressing MCF-7 cells (black bars) following 24 h of exposure to varying concentrations of drugs. Effects of SPINK1 were measured using the student's t-test with vector as control (*p < 0.005; **p < 0.05; ***p < 1E−05).

1. 5-FU (5-fluorouracil)

2. SAHA (suberoylanilide hydoramic acid)

3. VP16 (etoposide)

4. TAM (tamoxifen)

5. ADR (adriamycin)

Figure 8. Protein levels of apoptotic genes in the presence of SPINK1

1. Protein levels of main apoptotic regulators were measured via Western blot 2 h after SPINK1CM (SH9), vecCM or no CM was added to MCF-7 cells.

2. Expression of main apoptotic regulators was measured in MCF-7 cells that overexpress SPINK1 at varying levels (SH5 and SH9), along with empty vector overexpression as a control.

Figure 9. Induction of resistance to chemically induced apoptosis

Parental MCF-7 cells were pretreated with vecCM (white bars), WT SPINK1CM (black bars) or mutant K18YCM and then exposed to drugs for 24 h. Apoptosis was measured via activated PARP (left panels) and activated caspase-3 (right panels) levels. (*p < 0.005; **p < 0.05; ***p < 1E−05).

1. 5-FU

2. SAHA

Figure 10. SPINK1 induces cellular invasion in vitro and in vivo

1. Cell invasion assay with MDA-MB-231 cells. Loss of invasion upon SPINK1 siRNA treatment, and rescued with SPINK1-CM.

2. Effect of SPINK1-CM on invasion in two breast cell lines. The total number of cells that invaded was measured after 24 h (MB231) or 48 h (BT549) in response to SPINK1-CM and vector control CM.

3. Effect of SF9spink1CM and immunoneutralization on MDA-MB-231 cells. (White bars—SF9vecCM; black bars—SF9spink1CM diluted 10×; checkered bars—CM with addition of neutralizing antibody against SPINK1).

4. Invasion of MDA-MB-231 treated with WT SPINK1 and mutant SPINK1. Number of cells invaded were measured 24 h after assay setup. (*p < 0.005; **p < 0.05).

Figure 11. SPINK1 enhances metastases in vivo

MDA-MB-231 vector-control cells or SPINK1-overexpressing cells were injected intravenously into three mice each, and total lung metastases were counted 12 weeks later. (*p < 0.005).