Wnt signaling dynamics in head and neck squamous cell cancer tumor-stroma interactions

Abstract

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Keywords: Sox2; Wnt signaling; Wnt3a; cancer stem cells; head and neck squamous cell cancer.

Figure 1.. Wnt signaling in HNSCC tumors and cell lines.

(A) HNSCC patient tissue was stained (IHC) for Axin2. Eight of the highest ten Axin2 expressing tumors had advanced TNM stage (≥TXN2 or M1); the overall trend was not significant. (‡= HNSCC cell line cases, *= PDX cases used in efficacy studies). Wnt3a, β-catenin, and Axin2 staining intensity is consistently highest at the tumor-stroma interface in HNSCC patient samples (Top= 10X, Bottom with border= 40X). (B) Western blots confirm Wnt3a protein overexpression in all matched cancer and CAF cell lines. (C) 013C^Wnt3a cells activated the TOP-Flash reporter, which was suppressed by the addition of Wnt inhibitors (OMP-18R5, OMP-54F28). Activation of TOP-Flash by 036C^Wnt3a and 067C^Wnt3a cells was both low and variable (not shown). (D) Co-culture of TOP-GFP (reporter) cancer cells with those expressing Wnt3a. Cells were co-cultured at three different ratios (1 Cancer^TOP-GFP:1 Cancer^Wnt3a, 1:4 and 1:10) in order to test different ligand availability. With co-culture, Wnt activity (GFP-positive cells) was observed in 013C and 036C cells. GFP was not detectable in 067C no matter the ratio or number of days of incubation. GFP-positive cells were quantified per microscope viewing field due to equivalent cell densities. (E) CAFs were co-cultured with their respective cancer pairs and similarly seeded at the three different ratios stated above. Using this co-culturing method, we show paracrine signaling wherein Wnt3a overexpressing CAFs can activate Wnt signaling in cancer cells containing the TOP-GFP reporter for all 3 cancer cells. GFP positive cells were quantified per view. *= P≤ .05, **= P≤ .01.

Figure 2.. The Wnt3a ligand also activates Wnt signaling in HNSCC CAFs.

(A) Wnt activity was not detectable by the TOP-Flash reporter in 067CAFs and 013CAFs, with some luciferase activity observed in 036CAFs. (B) Wnt3a overexpressing cells (red/top cell line), either CAFs or cancer cells, were co-cultured with CAFs containing the TOP-GFP reporter (green/bottom cell line). Wnt3a expression effectively activated Wnt signaling (TOP-GFP) in adjacent CAFs. The TOP-GFP assay was quantified by counting GFP-positive cells per view due to equivalent cell densities. All three CAF cell lines had increased GFP-positive cells when co-cultured with Wnt3a expressing cells (cancer or CAF). *= P≤ .05, **= P≤ .01.

Figure 3.. Preferential Wnt activation of cancer cells by Wnt3a.

(A) Elapsed time to activation of TOP-GFP in 013C and 013CAF cells following exposure to rWnt3a. Cells left of the dashed lines were seeded alone while all assayed cells to the right of the dashed line were seeded at a 1:1 ratio of cancer:CAFs. (B) Percentage of 013C and 13CAF cells that are TOP-GFP-positive at 72 hours following addition of rWnt3a. Cells left of the dashed line were seeded alone while all assayed cells to the right of the dashed line were seeded at a 1:1 ratio of cancer:CAFs. (C) Change in the percentage of GFP-positive cells in cultures containing inhibitors OMP-18R5 and OMP54F-28 compared to controls (Fig. 3B). (D) Elapsed time to activation of TOP-GFP for 013C and 013CAF cells following transfer of media from either 013C^Wnt3a or 013CAF^Wnt3a. Cells left of the dashed line were seeded alone while all assayed cells to the right of the dashed line were seeded at a 1:1 ratio of cancer:CAFs. (E) Percentage of 013C and 013CAF cells that are TOP-GFP-positive at 72 hours following media transfers from cancer cells or CAFs expressing Wnt3a. Cells left of the dashed line were seeded alone while all assayed cells to the right of the dashed line were seeded at a 1:1 ratio of cancer:CAFs. (F) Change in the percentage of GFP-positive cells in cultures following media transfer and with OMP-18R5 and OMP54F-28 compared to controls (Fig. 3E). *= P≤ .05, **= P≤ .01.

Figure 4.. Wnt3a primes cancer cells for invasion that is initiated in the presence of CAFs.

(A) Schemata of the modified Matrigel invasion assay design. Cancer cells were seeded in the insert well of Matrigel invasion chambers while CAFs were seeded on the bottom well as a chemoattractant. Media containing 10% FBS was used a baseline control. (B) Invasion was decreased in 013C^Wnt3a cells when compared to control 013C^Empty cells at baseline, but the addition of CAFs as chemoattractant increased the invasiveness of all cancer cells. Importantly, 013C^Wnt3a were more greatly affected by the addition of CAFs compared to control 013C^Empty cells. Invasiveness was increased in 067C^Wnt3a at baseline. CAFs as a chemoattractant decreased invasion by 067C^Empty3acontrol cells. However, this decrease was “rescued” with the addition of CAFs in 067C cells expressing Wnt3a (067^Wnt3a.). Neither Wnt3a or CAFs altered the invasiveness of 036C cells. (C) Protein expression of non-invasive and invasive cells (scraped from the bottom of the Matrigel membrane) with varying chemoattractant (Media, CAF-Empty, CAF-Wnt3a). *= P≤ .05, **= P≤ .01.

Figure 5.. Wnt3a expression and Wnt signaling pathway activation increases characteristics of CSCs.

(A) Exogenous Wnt3a expression significantly increases expression of CSC-related (Sox2, Oct4, Nanog), Wnt-related (Axin2, Nfatc2), and EMT-related (Snai2 genes in 013C cells. (B) Oct4 and Sox2 protein levels are increased in sphere cultures of 013C^Wnt3a cells on days four and seven respectively. (C) Wnt pathway activation significantly increases the ALDH⁺ population in both monolayer and sphere cultures of 013C. (D and E) Wnt3a expression in increases sphere formation in 013C. No significant change in sphere size or number was observed in 067C or 036C cells expressing Wnt3a, even though Wnt3a forced expression also activated TOP-GFP 036C cells as previously described. *= P≤ .05, **= P≤ .01.

Figure 6.. Wnt inhibition blocks HNSCC PDX tumor proliferation in vivo.

(A) Mice bearing HNSCC PDX tumors were treated every two weeks with Wnt inhibitors OMP-18R5 and OMP-54F28. Control tumors were harvested upon reaching 2,000mm³. OMP-18R5 significantly inhibited tumor growth in all four PDX models, while OMP-54F24 significantly inhibited growth in only CUHN013 and CUHN044. (B) Decreased Wnt3a and Axin2 staining in OMP-18R5 treated PDX tumors by IHC and H-score analysis. (C) Pretreatment of recipient mice with the Wnt ligand scavenger OMP-54F28, compared to PBS control animals, significantly (P=.048) blocked tumor growth by CSCs sorted from PDX tumors. (D) Conceptual model of cancer-TME interaction and its role in tumor progression. *= P≤ .05, **= P≤ .01.