Research ArticleCancer

Therapeutic Targeting of SPINK1-Positive Prostate Cancer

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Science Translational Medicine  02 Mar 2011:
Vol. 3, Issue 72, pp. 72ra17
DOI: 10.1126/scitranslmed.3001498

Abstract

Gene fusions involving ETS (erythroblastosis virus E26 transformation–specific) family transcription factors are found in ~50% of prostate cancers and as such can be used as a basis for the molecular subclassification of prostate cancer. Previously, we showed that marked overexpression of SPINK1 (serine peptidase inhibitor, Kazal type 1), which encodes a secreted serine protease inhibitor, defines an aggressive molecular subtype of ETS fusion–negative prostate cancers (SPINK1+/ETS, ~10% of all prostate cancers). Here, we examined the potential of SPINK1 as an extracellular therapeutic target in prostate cancer. Recombinant SPINK1 protein (rSPINK1) stimulated cell proliferation in benign RWPE as well as cancerous prostate cells. Indeed, RWPE cells treated with either rSPINK1 or conditioned medium from 22RV1 prostate cancer cells (SPINK1+/ETS) significantly increased cell invasion and intravasation when compared with untreated cells. In contrast, knockdown of SPINK1 in 22RV1 cells inhibited cell proliferation, cell invasion, and tumor growth in xenograft assays. 22RV1 cell proliferation, invasion, and intravasation were attenuated by a monoclonal antibody (mAb) to SPINK1 as well. We also demonstrated that SPINK1 partially mediated its neoplastic effects through interaction with the epidermal growth factor receptor (EGFR). Administration of antibodies to SPINK1 or EGFR (cetuximab) in mice bearing 22RV1 xenografts attenuated tumor growth by more than 60 and 40%, respectively, or ~75% when combined, without affecting PC3 xenograft (SPINK1/ETS) growth. Thus, this study suggests that SPINK1 may be a therapeutic target in a subset of patients with SPINK1+/ETS prostate cancer. Our results provide a rationale for both the development of humanized mAbs to SPINK1 and evaluation of EGFR inhibition in SPINK1+/ETS prostate cancers.

Introduction

Therapies targeted against specific molecular alterations present only in cancer cells have revolutionized the treatment of several cancers. For example, targeting ERBB2, which is amplified in ~20% of breast cancers, with the humanized monoclonal antibody (mAb) trastuzumab (Herceptin) has resulted in improved survival for breast cancer patients. Although organ-confined prostate cancer is highly curable, more than 32,000 U.S. men are expected to die of metastatic prostate cancer in 2010 (1). Multiple approved therapies (and newer agents in late-stage development) target the androgen signaling axis in metastatic disease; however, additional targeted therapies are lacking.

We previously used a bioinformatics approach, cancer outlier profile analysis (COPA), to systematically prioritize genes with marked overexpression in a subset of cancers (outlier expression). This strategy identified outlier expression of the ETS (erythroblastosis virus E26 transformation–specific) family members ERG and ETV1 in a subset of prostate cancers across multiple gene expression profiling studies. It also led to the discovery of recurrent gene fusions involving the 5′ untranslated region of the androgen-regulated gene TMPRSS2 with ETS transcription factors (ERG, ETV1, ETV4, or ETV5) (25). Subsequent in vitro and in vivo studies have demonstrated a driving role for ETS fusions in prostate oncogenesis and cancer progression (69).

Subsequently, we used a “meta-outlier approach,” which used COPA to prioritize genes that consistently showed high-ranking outlier expression across multiple profiling studies. This approach identified SPINK1 (serine peptidase inhibitor, Kazal type 1) as a high-ranking meta-outlier in a subset of prostate cancer with mutually exclusive outlier expression of ERG and ETV1 across multiple prostate cancer profiling studies (10). SPINK1, also known as pancreatic secretory trypsin inhibitor (PSTI) or tumor-associated trypsin inhibitor (TATI), encodes a 56–amino acid peptide thought to protect the pancreas from autodigestion by preventing premature activation of pancreatic proteases (11). Apart from its normal expression in pancreatic acinar cells, SPINK1 mRNA has been reported to be expressed in various human cancers (1218), and increased serum SPINK1 concentration has been correlated with poor prognosis in some studies (12, 13, 17). The prostate gland also secretes a variety of serine proteases, most notably the kallikrein enzyme PSA (prostate-specific antigen), but also trypsin (19). Thus, SPINK1 may have a role in modulating the activity of cancer-related proteases in other tissues besides the pancreas.

We confirmed the mutually exclusive overexpression of SPINK1 and ETS gene fusions using a combined immunohistochemistry (for SPINK1) and fluorescence in situ hybridization (FISH) (for ETS fusions) approach across multiple independent cohorts, and demonstrated that SPINK1 outlier expression is associated with an aggressive subset of prostate cancers (10). We also demonstrated that SPINK1 outlier expression can be detected noninvasively in urine and contributes to a multiplexed panel of biomarkers, which outperforms serum PSA for prostate cancer diagnosis in patients presenting for needle biopsy (10, 20). Our combined analyses of more than 1500 prostate cancer cases demonstrated SPINK1 outlier expression in ~10% of all PSA-screened prostate cancers, which were invariably negative for ETS gene fusions (SPINK1+/ETS) (10). Furthermore, SPINK1+ tumors show shorter PSA recurrence-free survival in prostatectomy-treated patients (10) and shorter progression-free survival in endocrine-treated patients (21).

Unlike ETS gene fusions that lead to the overexpression of a transcription factor (which are difficult to target therapeutically), SPINK1 encodes an extracellular secreted protein and thus is potentially more amenable to therapeutic targeting. Here, we qualify SPINK1 as a therapeutic target in SPINK1+/ETS prostate cancer and demonstrate the therapeutic potential of a mAb to SPINK1 in preclinical models. Additionally, we demonstrate that SPINK1 mediates its oncogenic effects in part through epidermal growth factor receptor (EGFR) and that a mAb to EGFR shows in vitro and in vivo activity in SPINK1+ prostate cancer.

Results

SPINK1 as an autocrine factor in prostate cancer

To further investigate the role of SPINK1 in prostate cancer, we determined the effects of exogenous SPINK1 on invasion and proliferation using recombinant hexahistidine (6XHis)–tagged SPINK1 protein (rSPINK1) (fig. S1A) or conditioned media (CM) collected from 22RV1 prostate cancer cells (SPINK1+/ETS) (fig. S1B) (10). We treated benign immortalized RWPE prostate epithelial cells and DU145 and PC3 prostate cancer cells (both of which are SPINK1/ETS) with rSPINK1 (10 ng/ml), which resulted in a significant increase in cell proliferation (Fig. 1A). We next characterized the effect of rSPINK1 or 22RV1 CM on cell invasion using a Boyden chamber Matrigel invasion assay. As shown in Fig. 1B, addition of rSPINK1 or 22RV1 CM to RWPE cells significantly increased invasion (P = 0.003 and 0.0009, respectively). Similar effects were observed when MCF7 breast cancer cells were treated with rSPINK1 or 22RV1 CM (fig. S1C). Multiple recombinant 6XHis-tagged control proteins or CM collected from RWPE or LNCaP prostate cancer cells did not induce invasion in RWPE cells (figs. S1D and S2).

Fig. 1

SPINK1 has oncogenic effects in prostate cells in vitro. (A) SPINK1 stimulated cell proliferation in SPINK1/ETS cell lines. Benign immortalized prostate cell line RWPE and prostate cancer cell lines DU145 and PC3 (all SPINK1/ETS) were untreated or treated with rSPINK1 (10 ng/ml). Cell proliferation was measured by a WST-1 colorimetric assay at the indicated time points. (B) SPINK1 mediates invasion of RWPE cells as measured by Boyden chamber Matrigel invasion assay. RWPE cells were treated with rSPINK1 (10 ng/ml) or conditioned media (CM) from 22RV1 cells (SPINK1+/ETS). (C) As in (B), except using 22RV1 cells transfected with siRNA against SPINK1. SPINK1-silenced 22RV1 cells were further treated with rSPINK1 (10 ng/ml) or CM from 22RV1 cells. (D) SPINK1 expression in SPINK1 knockdown 22RV1 cells (stable pooled shSPINK1 or stable shSPINK1 clone 11) compared to nontargeting pooled stable control (shNS vector) cells by qPCR (transcript) or immunofluorescence using an antibody against SPINK1 (protein, upper inset; 600× magnification). (E) Invasion assay using shSPINK1 and shNS cells. Representative photomicrographs (400× magnification) showing cell motility assay (top inset) are shown. shNS vector cells exhibit longer cell motility tracks compared to shSPINK1 knockdown cells. (F) Cell proliferation assay using pooled shSPINK1, shSPINK1 clone 11, or shNS cells at the indicated time points. (G) Soft agar colony assay using pooled shSPINK1 and shNS cells. All experiments were independently performed in triplicate. Data represent means ± SEM. P values from significant two-sided Student’s t tests are given (*P < 0.05; **P < 0.001).

We previously showed that transient small interfering RNA (siRNA)–mediated knockdown of SPINK1 in 22RV1 cells decreased cell invasion (10). Here, we extended these results by demonstrating that the addition of rSPINK1 or 22RV1 CM rescued the invasive phenotype of 22RV1 cells in which SPINK1 was knocked down (Fig. 1C; P = 0.001 for both rSPINK1 and 22RV1 CM).

We next investigated whether the exogenous effect of SPINK1 on cell proliferation and invasion was dependent on protease inhibitory activity of trypsin [which has been shown to be simultaneously expressed with SPINK1 in different tumor types (17, 22)] or PSA. Initial experiments demonstrated that PRSS1 (trypsinogen) mRNA expression in 22RV1 cells is relatively low compared with the CAPAN-1 pancreatic cancer cell line (fig. S3A), although a significant increase in PRSS1 transcript was observed in siRNA-mediated SPINK1 knockdown 22RV1 cells (fig. S3B). However, as shown in fig. S3C, stimulation of 22RV1 cells with rSPINK1 or EGF did not affect trypsin expression. siRNA-mediated knockdown of PRSS1 in 22RV1 cells also had no effect on invasion (fig. S3, D and E). Similarly, stimulation of 22RV1 cells with rSPINK1 or EGF did not significantly affect PSA expression (fig. S4A). Finally, blocking PSA with a mAb did not significantly inhibit 22RV1 cell invasion (fig. S4B). Together, these findings demonstrate that extracellular SPINK1 induces prostate cancer cell proliferation and invasion independent of protease inhibitory activity of trypsin or PSA. Although effects on other proteases cannot be excluded, our results suggest that SPINK1 is an autocrine pro-proliferative and proinvasive factor with effects independent of trypsin and PSA activity.

The role of SPINK1 in cell proliferation and invasion

To further investigate the role of SPINK1 in cell proliferation and invasion, we generated short hairpin RNA (shRNA) against SPINK1 and established stable 22RV1 cells where SPINK1 was silenced (shSPINK1). Knockdown of SPINK1 in both pooled and clonal shSPINK1 cells compared to nontargeting control cells (shNS cells) was confirmed at the RNA level by quantitative polymerase chain reaction (qPCR) (more than 80% in both), as well as at the protein level by immunofluorescence staining with an antibody against SPINK1 (Fig. 1D). Next, we investigated the role of SPINK1 in cell invasion and motility using shSPINK1 cells. As anticipated, shSPINK1 cells showed decreased cell invasion by more than 75% in a Boyden chamber Matrigel assay compared to nonspecific vector control (shNS) cells (Fig. 1E; P = 0.002). Reduction of cell motility in a bead motility assay was also observed in shSPINK1 cells compared to shNS cells (Fig. 1E, top panel).

To investigate the role of SPINK1 in cell proliferation, we carried out assays using pooled shSPINK1, the clone with the greatest SPINK1 knockdown (shSPINK1 clone 11), and shNS cells. Both pooled (55% reduction) and clonal shSPINK1 cells (66% reduction) showed significantly decreased proliferation compared to shNS cells (Fig. 1F; P = 0.00002 in both cases). Further, shSPINK1 cells showed decreased soft agar colony formation when compared to shNS cells (Fig. 1G).

In vitro targeting of SPINK1 using a mAb

Because our results above demonstrate a role for SPINK1 in invasion and proliferation, and SPINK1 is an extracellular secreted protein, we hypothesized that a mAb against SPINK1 may be able to directly target SPINK1+/ETS prostate cancer cells. Thus, we tested the effects of an antibody to SPINK1 on 22RV1 cell proliferation and invasion. The SPINK1 mAb (0.5 and 1 μg/ml) significantly inhibited 22RV1 cell proliferation by 40 and 50%, respectively, compared to a control monoclonal immunoglobulin G (IgG) antibody (Fig. 2, A and B; P = 0.0001 and P = 0.0007, respectively). However, the antibody to SPINK1 had no effect on DU145 and PC3 cell proliferation.

Fig. 2

An antibody to SPINK1 attenuates in vitro proliferation and invasion exclusively in SPINK1+/ETS prostate cancer cells. (A) Cell proliferation of DU145, PC3, and 22RV1 cells was assessed in the presence of SPINK1 mAb or IgG mAb (1 μg/ml). (B) As in (A), except using 22RV1 cells and SPINK1 mAb or IgG mAb (0.5 to 1 μg/ml). (C) Effect of SPINK1 mAb or IgG mAb on invasion of SPINK1+/ETS cells (22RV1 and CWR22Pc) and SPINK1+/ETS cells (DU145, PC3, LNCaP, and VCaP). All experiments were independently performed in triplicates. Data represent means ± SEM. P values from significant two-sided Student’s t tests are given (*P < 0.05; **P < 0.001).

In addition to inhibiting proliferation, the mAb to SPINK1 (0.5 and 1 μg/ml) significantly attenuated cell invasion by 69 and 81%, respectively, compared to a control IgG mAb in 22RV1 cells (Fig. 2C; P = 0.002 and P = 0.007, respectively). Similar to 22RV1, which is an androgen signaling–independent derivative of primary CWR22 human prostate xenograft tumors, we also investigated CWR22Pc cells, an androgen signaling–dependent derivative of CWR22 (23), which also express high amounts of SPINK1. As expected, CWR22Pc cell invasion was blocked by 47 and 54% by the mAb to SPINK1 at 0.5 and 1 μg/ml of SPINK1 mAb concentration (Fig. 2C; P = 0.003 and P = 0.002, respectively). The mAb to SPINK1 had no significant effect on invasion of SPINK1 prostate cancer cell lines including PC3, DU145, LNCaP, or VCaP (Fig. 2C). Finally, the mAb to SPINK1 attenuated 22RV1 cell motility compared to IgG control, but had no effect on PC3 (SPINK1/ETS) cell motility (fig. S5A).

Oncogenic effects of SPINK1 in part through interaction with EGFR

SPINK1 has a similar structure as EGF, with ~50% sequence homology and three intrachain disulfide bridges (24, 25). To characterize potential SPINK1 and EGFR interaction, we overexpressed EGFR in human embryonic kidney (HEK) 293 cells and incubated the lysates with SPINK1-GST (glutathione S-transferase), GST, or GST-VEGF (vascular endothelial growth factor) receptor 2 (GST-VEGFR) recombinant proteins. We observed a strong interaction between SPINK1-GST and EGFR but not with GST alone or GST-VEGFR recombinant protein (Fig. 3A, top panel). Endogenous SPINK1 and EGFR interaction was not detected by immunoprecipitation and immunoblotting in 22RV1 cells, because of the secretory nature of the SPINK1 protein. However, addition of GST-SPINK1 to 22RV1 cells followed by immunoprecipitation and immunoblotting confirmed the interaction of SPINK1 and endogenous EGFR in 22RV1 cells (Fig. 3A, bottom panel).

Fig. 3

SPINK1 mediates its oncogenic effects in part through EGFR. (A) Immunoprecipitation using antibodies to IgG, SPINK1, or GST of exogenous SPINK1-GST, GST, or GST-VEGFR added to HEK 293 cells transfected with EGFR and immunoblotted with an antibody to EGFR (top panel), and immunoprecipitation using antibodies to IgG or SPINK1 of exogenous SPINK1-GST added to 22RV1 cells and immunoblotted with an antibody to EGFR (bottom panel). (B) Western blot showing EGFR phosphorylation in response to rSPINK1 (100 ng/ml) or EGF (10 ng/ml) stimulation. (C) Invasion assay showing siRNA-mediated EGFR knockdown 22RV1 cells treated with rSPINK1 (10 ng/ml). (D) Same as in (C), except with RWPE cells. (E) Invasion assay showing rSPINK1 (10 ng/ml)–stimulated RWPE cells in the presence or absence of C225 [cetuximab (50 μg/ml)] or IgG mAb (50 μg/ml). (F) Invasion assay showing the effect of IgG or C225 antibody on SPINK1+ and SPINK1 cancer cells. (G) As in (F), except 22RV1 cells were treated with a combination of antibodies to SPINK1 (1 μg/ml) and/or C225 (50 μg/ml). (H) Cell proliferation assay using the indicated cells in the presence of IgG mAb or C225. All experiments were independently performed in triplicates. Data represent means ± SEM. P values from significant two-sided Student’s t tests are given (*P < 0.05; **P < 0.001).

To further delineate the role of EGFR mediation of SPINK1 in prostate cancer, we next assessed whether exogenous SPINK1 was capable of inducing EGFR phosphorylation (similar to the cognate ligand EGF). Stimulating 22RV1 cells with rSPINK1 resulted in EGFR phosphorylation, although weaker than that observed with EGF (Fig. 3B). rSPINK1 stimulation resulted in sustained EGFR phosphorylation over a 90-min time course, whereas EGF resulted in strong EGFR phosphorylation, which diminished after only 10 min. Similarly, stable shSPINK1 knockdown 22RV1 cells (pooled and clonal) showed decreased phosphorylated EGFR (pEGFR), with slightly decreased total EGFR (possibly because of EGFR degradation) (fig. S6A). Finally, we demonstrate that rSPINK1 is able to induce dimerization of EGFR, although more weakly than EGF (fig. S6B).

We next examined the functional consequences of SPINK1-EGFR interaction in the context of SPINK1+ prostate cancer using 22RV1 cells. Transient knockdown of EGFR (fig. S5B) blocked 22RV1 cell invasion by 75% (Fig. 3C; P = 0.004), which was partially rescued by addition of exogenous SPINK1. A similar effect of EGFR knockdown was observed in RWPE cells treated with rSPINK1 (Fig. 3D; P = 0.014 and P = 0.021, respectively). These results suggest that some but not all of SPINK1’s effects are mediated by EGFR.

Because mAbs to EGFR are Food and Drug Administration (FDA)–approved for certain cancers, we sought to determine whether EGFR blockade could inhibit the oncogenic effects of SPINK1. We first demonstrated that mAb to EGFR (cetuximab, C225) blocked the cell-invasive effects of rSPINK1 in RWPE cells (Fig. 3E). C225 also blocked cell invasion of SPINK1+ 22RV1 cells but not in SPINK1 cell lines DU145, PC3, LNCaP, or VCaP (Fig. 3F). Combining mAbs to SPINK1 and EGFR had an additive effect in the inhibition of 22RV1 cell invasion (Fig. 3G; P = 0.001). In contrast to mAb to SPINK1 (Fig. 2A), C225 had no effect on 22RV1 cell proliferation or PC3 and DU145 cell proliferation (Fig. 3H). Together, these experiments suggest that SPINK1 has both EGFR-dependent and EGFR-independent functions in prostate cancer.

As a preliminary exploration of the downstream signaling pathways involved in the SPINK1-EGFR axis, we studied the mitogen-activated protein kinase (MAPK) and protein kinase B/AKT pathways in stable SPINK1 knockdown 22RV1 cells (shSPINK1 clone 11). We observed decreased pMEK (phosphorylated mitogen-activated or extracellular signal–regulated protein kinase kinase), pERK (phosphorylated extracellular signal–regulated kinase), and pAKT (phosphorylated AKT) in stable shSPINK1 cells compared to control shNS cells (fig. S5C). Likewise, 22RV1 cells treated with SPINK1 mAb antibody showed decreased pERK (fig. S5D). These observations provide the foundation for further studies of the SPINK1-EGFR axis.

The role of SPINK1 in vivo and as a therapeutic target

Our in vitro studies demonstrated that SPINK1 mediates cell proliferation and invasion in SPINK1+ prostate cancer cells, and suggested that a mAb can target extracellular SPINK1. To investigate the role of SPINK1 in intravasation, a key step involved in the process of metastasis, we used a chick chorioallantoic membrane (CAM) model system (26) and demonstrate that rSPINK1 induced intravasation of benign RWPE cells (Fig. 4A). Similarly, SPINK1 mAb and C225 significantly inhibited 22RV1 cell intravasation (P = 0.01 and P = 0.03, respectively), but did not significantly inhibit PC3 cell intravasation (Fig. 4, B and C).

Fig. 4

SPINK1 is a therapeutic target in SPINK1+ prostate cancer. (A) Chick chorioallantoic membrane (CAM) assay quantifying intravasated RWPE cells upon stimulation with rSPINK1 (n = 6 in each group). (B) CAM assay using 22RV1 cells in the presence of IgG mAb, SPINK1 mAb, or C225 (n = 5 in each group), with fold change of intravasated cells compared to IgG mAb plotted. (C) As in (B), except using PC3 cells. (D) Subcutaneous xenograft growth of shNS-luciferase (luc) or shSPINK1-luc 22RV1 cells implanted in male BALB/c nu/nu mice (n = 10 in each group). (E) As in (D), except using 22RV1-luc cell xenografts treated with control IgG mAb (n = 8), SPINK1 mAb (n = 6), or C225 (n = 8) (10 mg/kg) twice a week. (F) Same as in (E), except mice (n = 7 per group) were treated with a combination of SPINK1 and C225 mAb (10 mg/kg for both). (G) As in (E) and (F), except using PC3-luc xenografts treated with control IgG mAb, SPINK1 mAb, or C225 (n = 8 per group) (10 mg/kg) alone or in combination twice a week. (H) Representative bioluminescence images from mice in (D) bearing pooled shNS-luc or shSPINK1-luc xenografts and percent reduction in tumor volume at week 5. (I) Same as (H), except bioluminescence images from mice bearing 22RV1-luc xenografts (red, top panel) or PC3-luc (blue, lower panel) mice treated with IgG mAb, SPINK1 mAb, or C225 mAb alone or in combination, with comparative percent reduction plot in tumor volume at week 5. Data represent means ± SEM. P values from significant two-sided Student’s t tests are given (*P < 0.05; **P < 0.001).

To qualify SPINK1 as a potential therapeutic target in vivo, we implanted pooled shSPINK1-luciferase (luc) and shNS-luc 22RV1 cells in nude male mice. At both 4 and 5 weeks after implantation, 22RV1-shSPINK1-luc cells formed significantly smaller tumors (55% reduction at week 4, P = 0.008, and 63% reduction at week 5, P = 0.013) compared to shNS-luc cells (Fig. 4, D and H).

To demonstrate preclinical efficacy of the mAb to SPINK1, we treated nude mice implanted with 22RV1-luc cells with either the mAb to SPINK1 or an isotype-matched monoclonal IgG (10 mg/kg) twice a week. As shown in Fig. 4, E and I, administration of SPINK1 mAb monotherapy resulted in a 61% reduction of tumor burden at week 4 (P = 0.015) and 58% reduction at week 5 (P = 0.015). A significant decrease in Ki-67–positive immunostained nuclei was observed in the SPINK1 mAb–treated group compared to the control group (fig. S7).

Because SPINK1 mediates its oncogenic effects in part through EGFR, we similarly assessed the mAb to EGFR (C225) using the same dosage schedule. C225 treatment resulted in a 41% reduction at week 4 (P = 0.04) and 37% reduction at week 5 (P = 0.02) (Fig. 4, E and I). By combining mAbs to SPINK1 and EGFR, we observed an additive effect in vivo showing a 74 and 73% reduction in the growth of 22RV1 xenografts at weeks 4 (P = 0.01) and 5 (P = 0.003), respectively (Fig. 4, F and I).

To confirm our in vitro results, which suggested no effect of SPINK1 or EGFR inhibition on SPINK1 prostate cancer, we performed a similar xenograft study using PC3 cells. As expected, neither SPINK1 mAb nor C225 significantly inhibited tumor growth in PC3 xenografted mice (Fig. 4, G and I). Finally, to investigate the potential toxicity of SPINK1 mAb therapy, we investigated whether the mAb to SPINK1 interacts with SPINK3, the murine homolog of SPINK1. The mAb to SPINK1 used in our studies does not recognize murine SPINK3, thus explaining the lack of observed toxicity in SPINK1 mAb–treated mice (fig. S8, A to C).

Discussion

Previous studies demonstrated that SPINK1 outlier expression identified a subset of ETS-negative prostate cancers (~10% of all PSA-screened prostate cancers), although the mechanism for SPINK1 outlier expression remains unknown (10). SPINK1 defines a distinct molecular subtype of prostate cancer characterized by lack of ETS gene fusions as well as a more aggressive phenotype as corroborated by independent groups across distinct cohorts of prostate cancer patients (10, 21). Thus, our working hypothesis is that SPINK1+ prostate cancer represents an aggressive form of prostate cancer that may respond to different therapies than ETS gene fusion–positive prostate cancers.

Here, we show that SPINK1 promotes prostate cancer proliferation and invasion through autocrine and paracrine signaling. We also demonstrate an in vivo role for SPINK1 in intravasation and tumor xenograft growth. At present, the precise mechanism and signaling pathways responsible for these effects in SPINK1+ prostate cancer are unclear. A recent study showed that mutation of SPINK1 at leucine 18 (L18) in the trypsin interaction site reduced tumor growth, angiogenesis, and lung metastases in HT-29 5M21 human colon carcinoma tumor xenografts, suggesting that the cancer-related phenotypes of SPINK1 may be related to its anti-proteinase activity (27). Moreover, the invasive behavior of these HT-29 5M21 colon cancer cells was abolished with an antibody to SPINK1 (27). However, in our study, we did not observe any effect of SPINK1 on trypsin or PSA, two candidate proteases in prostate cancer.

Recent studies also indicate that SPINK1 may be an apoptosis inhibitor preventing serine protease-dependent cell death (28). Here, we show that SPINK1, which has structural similarities with EGF (29), binds to EGFR, and inhibiting SPINK1 attenuates key downstream mediators of the EGFR pathway including MEK, ERK, and AKT. Furthermore, we also show that SPINK1 dimerizes EGFR and induces sustained phosphorylation of EGFR, which have been shown to be critical for downstream signaling activation after ligand binding (30). However, in contrast to SPINK1 mAb, EGFR mAb only partially inhibited the cell-invasive effects of 22RV1 cells and had no effect on cell proliferation, suggesting that SPINK1 engages both EGFR-dependent and EGFR-independent pathways to mediate its oncogenic effects. SPINK1 has also been shown to engage the EGFR/MAPK cascade in NIH 3T3 fibroblasts and pancreatic cancer cells (31).

This study provides compelling evidence that SPINK1 overexpression is oncogenic in prostate cancer and that inhibition of SPINK1 via RNA interference or blocking antibodies may have therapeutic potential. Our preclinical models suggest that this therapeutic effect would only be effective in patients with SPINK1+ prostate cancer, suggesting that such therapies would need to be evaluated in a molecularly guided fashion. Because the area of antibody-based therapeutics for extracellular targets is well developed, based on examples such as trastuzumab in breast cancers with ERBB2 overexpression, we postulate that a SPINK1-blocking antibody may have similar efficacy on a molecularly defined subset of prostate cancers. We have previously demonstrated that patients with the subset of SPINK1+/ETS prostate cancers can be reliably identified by immunohistochemistry (10, 20), as would be required for a molecularly defined clinical trial. Although humanized SPINK1 mAbs are not yet available for clinical testing, our studies show that SPINK1 partially mediates its oncogenic effects through EGFR.

This finding prompted us to evaluate the utility of the FDA-approved EGFR mAb cetuximab, which showed in vitro and in vivo activity only against SPINK1+ prostate cancer cells (although less effective than SPINK1 mAb). Phase I/II clinical trials of cetuximab (32) and EGFR small molecules have been largely disappointing in metastatic prostate cancer (33, 34); however, a small subset of patients have had responses, including 3 of 36 (8%) patients who showed >50% PSA decline in a Phase Ib/IIa clinical trial of cetuximab in combination with doxorubicin in castrate-resistant metastatic prostate cancer patients (32). Results from our study provide a plausible mechanism for why only the limited subset of patients with positive cancers (~10% of all cases) may benefit from EGFR inhibition. This hypothesis can be assessed retrospectively and in biomarker-informed clinical trials of patients with SPINK1+ prostate cancer. Because the mAb to SPINK1 used in our studies did not interact with murine SPINK3 (the homolog of SPINK1), our study does not inform on the potential toxicity of SPINK1 mAb therapy. However, an FDA-approved mAb to EGFR has specific in vivo activity against SPINK1+ prostate cancer, providing an immediately translatable strategy for targeting SPINK1+ cancers that can be clinically investigated while toxicity of humanized SPINK1 antibody therapy is explored.

In summary, our results support SPINK1 as an oncogene in a subset of prostate cancers that can be molecularly identified, and provide the rationale to develop humanized SPINK1 antibodies for human clinical trials. Our work also reinforces the molecular subclassification of prostate cancer in clinical trials (whether through SPINK/ETS status or other relevant biomarkers), which has lagged behind other common epithelial cancers (that is, breast, lung, and colon).

Materials and Methods

Cell lines and SPINK1 knockdown

The benign immortalized prostate cell line RWPE as well as prostate cancer cell lines DU145, PC3, and 22RV1 were obtained from the American Type Culture Collection (ATCC) and were grown according to ATCC guidelines. For stable knockdown of SPINK1, human lentiviral shRNAmir individual clone (ID V2LHS_153419) targeting against SPINK1 or nonsilencing lentiviral shRNAmir in GIPZ vectors was purchased from Open Biosystems (Thermo Scientific Open Biosystems). Details are available in Supplementary Materials and Methods.

Quantitative PCR

Total RNA was isolated with a miRNeasy mini kit following the manufacturer’s instruction (Qiagen). Complementary DNA was synthesized from 1 μg of total RNA with SuperScript III (Invitrogen) in the presence of random primers. qPCR was performed with the StepOne Real-Time PCR system (Applied Biosystems). Details and primer information are available in Supplementary Materials and Methods.

Cell proliferation assay

Proliferation for control and experimental cells was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases (cell proliferation reagent WST-1; Roche Diagnostics) at the indicated time points in triplicate. Cell counts for shNS vector and shSPINK1 cells were estimated by trypsinizing cells and analysis by Coulter counter (Beckman Coulter) at different time points in triplicates.

Basement membrane matrix invasion assay

For invasion assays, shNS vector– or shSPINK1-transduced cells, as well as RWPE, PC3, and 22RV1 cells were used. Equal numbers of the indicated cells were seeded onto the basement membrane matrix (BD Biosciences) present in the insert of a 24-well culture plate. RPMI media supplemented with 10% fetal bovine serum were added to the lower chamber as a chemoattractant. After 48 hours, noninvading cells and extracellular matrix were removed with a cotton swab. Invaded cells were stained with crystal violet and photographed. The inserts were treated with 10% acetic acid, and absorbance was measured at 560 nm.

CAM assay

The assay was performed essentially as described (26). Two million RWPE cells were mixed with either 200 ng of multiple tag control protein or 200 ng of rSPINK1 protein and applied to the CAM of 11-day-old chicken embryo. Similarly, 2 million 22RV1 or PC3 cells were mixed with either monoclonal IgG or antibodies to SPINK1 or C225 (1 μg/ml) and applied onto the upper CAM of a fertilized chicken embryo. Three days after implantation, the relative number of cells that intravasate into the vasculature of the lower CAM was analyzed by extracting genomic DNA with the Puregene DNA purification system. Quantification of the human cells in the extracted DNA was done as described (35).

22RV1 and PC3 xenograft models

Four-week-old male BALB/c nu/nu mice were purchased from Charles River Inc. (Charles River Laboratory). Stable 22RV1 shNS-luc and 22RV1 shSPINK1-luc cells (5 × 105), or 22RV1-luc (2 × 105) or PC3-luc (5 × 105) cells were resuspended in 100 μl of saline with 20% Matrigel (BD Biosciences) and were implanted subcutaneously into the left flank regions of the mice. Details are available in Supplementary Materials and Methods.

Statistical analysis

All values presented in the study were expressed as means ± SEM. The significant differences between the groups were analyzed by a Student’s t test, and a P value of <0.05 or <0.001 was considered significant.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/3/72/72ra17/DC1

Materials and Methods

Fig. S1. rSPINK1 or CM collected from 22RV1 cells induces invasion in benign or cancer cells.

Fig. S2. CM collected from 22RV1 cells induces cell invasion, but not CM, from LNCaP cells.

Fig. S3. PRSS1 (trypsin1) knockdown in 22RV1 cells has no effect on SPINK1-mediated cell invasion.

Fig. S4. Exogenous rSPINK1 has no effect on PSA in 22RV1 cells.

Fig. S5. SPINK1 mAb reduces SPINK1+ cell motility and SPINK1 knockdown alters MAPK pathway.

Fig. S6. Exogenous SPINK1 induces EGFR dimerization and phosphorylation.

Fig. S7. SPINK1 mAb induces decrease in tumor proliferation index.

Fig. S8. Anti-SPINK1 mAb, which does not recognize the murine homolog of SPINK1 (SPINK3), has no observed toxic effect in treated mice.

Reference

Footnotes

  • Citation: B. Ateeq, S. A. Tomlins, B. Laxman, I. A. Asangani, Q. Cao, X. Cao, Y. Li, X. Wang, F. Y. Feng, K. J. Pienta, S. Varambally, A. M. Chinnaiyan, Therapeutic Targeting of SPINK1-Positive Prostate Cancer. Sci. Transl. Med. 3, 72ra17 (2011).

References and Notes

  1. Acknowledgments: We thank X. Jiang, X. Jing, A. Yocum, J. Siddiqui, K. Suleman, R. Mehra, and C. A. Maher for the technical assistance; M. Dhanasekaran and C. Brenner for discussions; and J. Granger for critically reading the manuscript. Funding: This work is supported in part by the Department of Defense W81XWH-08-1-0031, Early Detection Research Network UO1 CA111275, Prostate SPORE P50CA69568, and NIH (R01CA132874). A.M.C. is supported by the Doris Duke Charitable Foundation Clinical Scientist Award, Burroughs Welcome Foundation Award in Clinical Translational Research, and the Prostate Cancer Foundation (PCF). A.M.C. is an American Cancer Society research professor. B.A. is supported by the Genentech Foundation Postdoctoral Fellowship and Young Investigator Award from the Expedition Inspiration Fund for Breast Cancer Research. S.A.T. is supported by a Young Investigator Award from the PCF. Q.C. is supported by U.S. Department of Defense (PC094725). S.V. is supported by a Prostate Cancer SPORE Career Development award. Author contributions: B.A., S.A.T., and A.M.C. designed the research plan and wrote the manuscript; B.A., B.L., Q.C., and X.C. performed the in vitro experiments; I.A.A. performed CAM assays; B.A. performed in vivo xenograft experiments; B.A., B.L., I.A.A., S.A.T., F.Y.F., K.J.P., S.V., and A.M.C. analyzed the data. Competing interests: The University of Michigan has filed for patents on SPINK1, on which A.M.C., B.A., and S.A.T. are named as inventors. A.M.C. is a consultant for Gen-Probe Inc. S.A.T. has consulted for Cougar Biotechnology, AstraZeneca, and Compendia Biosciences. The diagnostic field of use has been licensed to Gen-Probe Inc. Gen-Probe was not involved in the design or funding of these studies. The other authors declare that they have no competing interests.
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