Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma

Abstract

Background: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide.

Objectives: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC.

Methods: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s.

Results: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo.

Conclusions: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

Figure 1

Expression of C1r and C1s is upregulated in cutaneous squamous cell carcinoma (cSCC) cells. (a) C1R and C1S mRNA levels in normal human epidermal keratinocytes (NHEKs; n = 10) and cSCC cell lines (n = 8) were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR). RNA from human liver was used as a positive control. (b) The levels of C1r and C1s in NHEKs and cSCC cell lines were determined by Western blotting of conditioned media under nonreducing conditions (upper panel), with β‐actin in cell lysates as a sample control. C1r and C1s protein levels were quantitated densitometrically and corrected for β‐actin levels, and the ratio of C1s to C1r was calculated. The levels of the cleaved form of C1s in conditioned media of NHEKs and cSCC cell lines were determined by Western blotting under reducing conditions (lower panel). (c) Levels of C1R and C1S mRNA in normal skin (n = 10) and cSCC (n = 6) samples were analysed by qRT‐PCR. (d) Xenografts established with human cSCC cells were stained for immunohistochemistry using C1r and C1s antibodies. Scale bar = 100 μm. Statistical analysis was performed with Mann–Whitney two‐way U‐test. *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

Expression of C1r by tumour cells in cutaneous squamous cell carcinoma (cSCC). (a–f) Sections of tissue microarray blocks containing samples from ultraviolet‐induced sporadic cSCC (n = 106), recessive dystrophic epidermolysis bullosa‐associated cSCCs (RDEBSCC, n = 15), cSCC in situ (cSCCIS, n = 61), premalignant lesions (actinic keratosis, AK, n = 61) and normal skin (n = 8) were stained with C1r antibody. Strong cytoplasmic staining was detected in tumour cells in cSCC (a, b) and RDEBSCC (c). Staining for C1r was absent or weak in normal skin (d). In AK (e) and cSCCIS (f) staining was weak. Scale bar = 100 μm. (g) C1r immunostaining was scored as negative (−), weak (+), moderate (++) or strong (+++) based on the specific staining intensity. **P < 0·01, ***P < 0·001 by χ²‐test.

Figure 3

Expression of C1s by tumour cells in cutaneous squamous cell carcinoma (cSCC). (a–f) Sections of tissue microarray blocks containing samples from ultraviolet‐induced sporadic cSCC (n = 115), recessive dystrophic epidermolysis bullosa‐associated cSCCs (RDEBSCC, n = 16), cSCC in situ (cSCCIS, n = 63), premalignant lesions (actinic keratosis, AK, n = 57) and normal skin (n = 21) were stained with C1s antibody. Cytoplasmic staining for C1s was moderate (a) or strong (b) in cSCC and strong in RDEBSCC (c). In normal skin the staining for C1s was mainly weak (d). In AK (e) and cSCCIS (f) staining was weak or moderate. Scale bar = 100 μm. (g) C1s immunostaining was scored as negative (−), weak (+), moderate (++) or strong (+++) based on the specific staining intensity. *P < 0·05, ***P < 0·001 by χ²‐test.

Figure 4

Knockdown of C1r and C1s promotes apoptosis of cutaneous squamous cell carcinoma (cSCC) cells. (a, b) cSCC cells (UT‐SCC‐12A) were transfected with C1R small interfering (si)RNA (C1r siRNA_7 or C1r siRNA_11; 75 nmol L⁻¹) (a) or C1S siRNA (C1s siRNA_5; 120 nmol L⁻¹) (b) and control siRNA. Cell lysates were analysed by Western blotting 8 days after transfections. The levels of C1R and C1S were quantitated densitometrically and corrected for β‐actin levels in the same samples. (c, d) cSCC cells (UT‐SCC‐12A) were transfected with control siRNA, C1R (75 nmol L⁻¹) (c) or C1S siRNAs (120 nmol L⁻¹) (d). The confluency of the cells was determined at the indicated time points using IncuCyte ZOOM (n = 6–8; mean ± SEM). (e–g) UT‐SCC‐12A cells were transfected with C1R (75 nmol L⁻¹) or C1S (120 nmol L⁻¹) siRNAs or control siRNA; 48 h after transfection apoptotic cells were detected with TUNEL staining, and the relative number of TUNEL‐positive cells was counted (mean ± SEM) (g). Representative images after C1r (e) and C1s (f) knockdown are shown. Scale bar = 1 μm. *P < 0.05, **P < 0.01, ***P < 0.001 by t‐test

Figure 5

Knockdown of C1r and C1s inhibits migration of cutaneous squamous cell carcinoma (cSCC) cells. UT‐SCC‐12A cells were transfected with C1R (75 nmol L⁻¹) (a) or C1S (120 nmol L⁻¹) (c) small interfering (si)RNAs or control siRNA, and incubated for 48 h (n = 8) (mean ± SEM). A wound was made using 96‐well WoundMaker and incubation was continued in 0·5 mmol L⁻¹ hydroxycarbamide. Representative images after C1r (b) and C1s (d) knockdown are shown. Scale bar = 300 μm. *P < 0·05, **P < 0·01, ***P < 0·001 by t‐test.

Figure 6

Knockdown of C1r and C1s suppresses growth of cutaneous squamous cell carcinoma (cSCC) in vivo. (a) cSCC cells (UT‐SCC‐91) were transfected with C1R small interfering (si)RNA_5 (n = 7), C1S siRNA_5 (n = 8) (120 nmol L⁻¹) or control siRNA (n = 8) and incubated for 72 h. Cells (7 × 10⁶) were injected subcutaneously into the back of severe combined immunodeficient mice and the size of tumours was measured twice a week (mean ± SEM). (b) Xenografts were harvested after 16 days and stained with haematoxylin and eosin (HE), and for immunohistochemistry for the proliferation marker Ki‐67, the vascular endothelial marker CD34 and the apoptotic marker active caspase‐3, with Mayer's haematoxylin as counterstain. Representative stainings from each group are shown. Arrows indicate CD34‐positive blood vessels. Scale bar = 100 μm. (c–e) The percentage of Ki‐67‐positive cells (c), the number of CD34‐positive blood vessels (d) and the percentage of active caspase‐3‐positive cells (e) were counted. *P < 0·05, **P < 0·01, ***P < 0·001 by Mann–Whitney U‐test.

Figure 7

Alteration of the gene expression profile in cutaneous squamous cell carcinoma (cSCC) cells after C1s knockdown. cSCC cell lines (UT‐SCC‐12A, ‐59A and ‐91) were transfected with C1S small interfering (si)RNA_5 or control siRNA (120 nmol L⁻¹) and mRNA sequencing was performed 72 h after transfection. Summary of Ingenuity Pathway Analysis biofunctions, gene ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Reactome related to C1s knockdown (P < 0·05, fold change log₂ 1·0).

Figure 8

Alteration of the gene expression profile in cutaneous squamous cell carcinoma (cSCC) cells after C1s knockdown. (a) Significantly regulated genes belonging to the gene ontology term ‘negative regulation of MAPK cascade’ are shown in gene blot. (b) Significantly regulated genes belonging to the gene ontology term ‘phosphatidylinositol 3‐kinase activity’ are shown in gene blot.

Figure 9

Decreased activation of Akt and ERK1/2 in cutaneous squamous cell carcinoma (cSCC) cells after C1s knockdown. cSCC cells (UT‐SCC7) were transfected with C1S small interfering (si)RNAs and control siRNA (120 nmol L⁻¹) and the levels of phosphorylated Akt (p‐Akt), phosphorylated extracellular signal‐related kinase 1/2 (p‐ERK1/2), total Akt and total ERK1/2 were analysed by Western blotting 72 h after transfection. The levels of p‐Akt and p‐ERK1/2 in blots were determined densitometrically and corrected for the levels of total Akt and ERK1/2 in the same samples, respectively (values below the blots). β‐actin was used as loading control.