Targeting an autocrine IL-6-SPINK1 signaling axis to suppress metastatic spread in ovarian clear cell carcinoma

Abstract

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Figure 1.. SPINK1 knockdown sensitizes OCCC tumor cells to anoikis and inhibits proliferation.

(a-c) Significantly reduced survival of (a, b) JHOC9 and (c) JHOC5 cells when transduced with two different shRNA constructs targeting SPINK1 (KD1 and KD2) and grown on ultra-low attachment conditions, assessed by Annexin V and propidium iodide (PI) and quantified (b,c) by flow cytometry; data shown represent the mean and SE of three independent experiments. (One-way Anova). (d, e) Time course analysis of Annexin V binding to JHOC9 (d) and JHOC5 (e) cells with knockdown of SPINK1 (red) and rescue of knockdown by addition of 500 nM recombinant SPINK1 (blue) as compared to nontarget control (black); quantification at 4 h time point. Data represent mean and SD for triplicate wells; these experiments were independently repeated with confirmatory results shown in Fig. S4. (Unpaired t-test). (f-h) Decreased proliferation as assessed by EdU incorporation of JHOC9 (f,g) and JHOC5 (h) cells with SPINK1 knockdown; data shown represent the mean and SE of two independent experiments (One-way Anova). * p<0.05, ** p<0.01.

Figure 2.. SPINK1 knockdown in OCCC tumor cells reduces tumor burden and inhibits peritoneal metastasis.

(a, b) Time course of tumor growth of JHOC9 (a) and JHOC5 (b) cells injected IP into NOD/SCID mice assessed using bioluminescent imaging revealed significantly reduced tumor burden from week 7 and beyond for JHOC9 SPINK1 KD as compared to nontarget control (NT n=11, KD n=10), and from week 1 and beyond for JHOC5 SPINK1 KD as compared to nontarget control (NT n=5, KD n=6). (c-f) Reduced tumor spread with SPINK1 KD of JHOC 9 (c,d) and JHOC5 (e,f) cells at time of harvest (JHOC9, 15 weeks; JHOC5, 7 weeks), assessed through in vivo imaging prior to surgery (c,e top; quantified in d,f top) and ex vivo imaging of the body cavity (c,e bottom; quantified in d,f bottom). (g-j) Reduced acites volume (g,i) and reduced tumor presence in ascites and metastasis to omentum and peritoneum (h,j) with SPINK1 KD tumor mice for both JHOC9 (g,h) and JHOC5 (i,j) cells. (k.l) Scoring extracted tissue for tumor cell presence (any size lesion) showed reduced presence in multiple organs with SPINK1 KD for both JHOC9 (k) and JHOC5 (l) tumor models. Mann-Whitney test * p<0.05, ** p<0.01, *** p<0.001.

Figure 3.. The IL-6 signaling pathway drives SPINK1 expression in OCCC.

(a-c) JHOC9 (a), JHOC5 (b), and human OCCC tumors (c) stained for SPINK1, IL-6, IL6Rα, and gp130 in adjacent tissue sections demonstrated colocalization of SPINK1 expression with IL-6 pathway components (size bar =100μm). (d) JHOC9 cells transduced with lentiviral shRNA IL-6 knockdown constructs KD1 and KD2 showed significant reduction in IL-6 mRNA expression assessed by qRT/PCR. (e) Cells from (d) with knockdown of IL-6 also showed significant reduction of SPINK1 transcript expression (One-way Anova). (f) JHOC9 cells treated with 30 ng/ml recombinant human IL-6 for 48 h showed significantly increased SPINK1 transcript expression. Results assessed from triplicate wells (Unpaired t-test). (g, h) Conditioned media of JHOC9 cells (g) or JHOC5 cells (h) show significantly reduced SPINK1 protein concentration from cells transduced with SPINK1 or IL-6 knockdown constructs compared to nontarget control cells, as measured by ELISA. Results assessed in triplicate wells (One-way Anova). * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001.

Figure 4.. IL-6 increases tumor cell survival and tumor cell proliferation through SPINK1 expression.

(a-c) JHOC9 (a,b) and JHOC5 (c) cells treated with 30 ng/ml recombinant human IL-6 show significantly increased survival under attachment free conditions. (d-f) JHOC9 (d, e) and JHOC5 cells (f) with knockdown of IL-6 show significantly decreased survival relative to nontarget control cells under attachment-free conditions. Data shown represent the mean and SE of three independent experiments. (One-way Anova). (g, h) Time course analysis of Annexin V binding to JHOC9 (g) and JHOC5 (h) cells with knockdown of IL-6 (green) and rescue of knockdown by addition of 500 nM recombinant SPINK1 (blue) as compared to nontarget control (black); quantification at 4 h time point. Data shown represent mean and SD for triplicate wells; these experiments were independently repeated with confirmatory results shown in Fig. S10. (Unpaired t-test). (i-k) Decreased proliferation was found in JHOC9 (I,j) and JHOC5 (k) cells with IL-6 knockdown relative to nontarget controls as assessed by EdU incorporation; data shown represent the mean and SE of two independent experiments (One-way Anova). * p<0.05, ** p<0.01, *** p<0.001.

Figure 5.. Common gene expression patterns with knockdown of SPINK1 and IL-6.

(a) Transcripts regulated by SPINK1 knockdown show significant overlap (p=3.0E-16) with dataset of ovarian clear cell carcinoma vs benign tissue. (b) Transcripts regulated by SPINK1 knockdown and IL-6 knockdown show significant overlap (p=3.0E-55). (c) Heat map of 422 transcripts significantly regulated by SPINK1 knockdown and IL-6 knockdown (J9, JHOC9; J5, JHOC5; SP1 KD, SPINK1 knockdown; IL-6 KD, IL-6 knockdown). (d-g) Validation via qRT/PCR of common transcriptional alterations induced by SPINK1 knockdown and IL-6 knockdown. (d, f) BTG2 was consistently upregulated in both cell lines and in both SPINK1 KD and IL-6 KD. (e, g) RFK, FBXO28, and MASTL showed consistent reduced expression with both SPINK1 KD and IL-6 KD in both cell lines. qRT/PCR analysis was conducted in triplicate wells. One-way Anova ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 6.. Tocilizumab targets IL-6 signaling to reduce SPINK1 expression, tumor cell survival, ascites accumulation and metastasis.

(a,b) JHOC9 cells treated for 48 h with tocilizumab, which targets IL-6Rα, showed decreased phosphorylation of downstream mediator STAT3 (b, quantification representing the mean and SE from two independent lysates, one-way Anova). (c) SPINK1 mRNA expression levels were significantly reduced after tocilizumab treatment (One-way Anova and unpaired t-test). (d, e) JHOC9 cells treated for 48 h with tocilizumab showed significantly reduced cell survival as assessed by Annexin V and PI staining (e, quantification represents the mean and SE of three independent experiments, unpaired t-test). (f-m) Tocilizumab treatment of JHOC9 (f-i) and JHOC5 (j-m) tumor models resulted in decreased ascitic fluid (g,k) and reduced metastasis to diaphragm (h,l) and omentum (i,m), as compared to IgG control. (Unpaired t-test), ** p<0.01, *** p<0.001.