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. 2015 Jun 24:13:199.
doi: 10.1186/s12967-015-0555-4.

The prognostic significance of CXCL1 hypersecretion by human colorectal cancer epithelia and myofibroblasts

Anne-France le Rolle  1 , Thang K Chiu  2 , Michael Fara  3 , Jinru Shia  4 , Zhaoshi Zeng  5 , Martin R Weiser  6 , Philip B Paty  7 , Vi K Chiu  8
Affiliations

The prognostic significance of CXCL1 hypersecretion by human colorectal cancer epithelia and myofibroblasts

Anne-France le Rolle et al. J Transl Med. .

Abstract

Background: Clinical therapy for metastatic colorectal cancer (CRC) remains limited, especially when the tumor harbors a KRAS mutation. This study aimed to identify prognostic biomarkers in CRC that are accessible for therapeutic inhibition.

Methods: Conditioned media from human CRC epithelial cells and myofibroblasts were screened by cytokine arrays for tumorigenic factors. The protein and mRNA expressions of these factors were determined by immunohistochemistry and gene microarrays in human CRC tissues. Prognostic biomarkers were determined by correlation of mRNA expression to overall survival in stage IV CRC patients. Inhibition of CXCL1 was performed with specific neutralizing antibody and lentiviral shRNAs. Malignant growth was assessed by soft agar growth assays and xenograft tumor growth in immunocompromised mice.

Results: CXCL1 was highly secreted by KRAS mutant human CRC cells and myofibroblasts in a complementary adaptive response to serum deprivation. Elevated CXCL1 level promoted anchorage-independent growth of murine fibroblasts and human CRC cells. Inhibition of CXCL1 by neutralizing antibody and specific shRNAs decreased CRC tumor growth. Highly elevated CXCL1 expression significantly correlated with decreased overall survival in stage IV CRC patients (hazard ratio 0.28; 95% CI 0.11-0.72).

Conclusions: High CXCL1 expression is a poor prognostic biomarker in metastatic CRC. CXCL1 inhibition suppressed tumorigenic growth of KRAS mutant CRC cells.

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Figures

Figure 1
Figure 1
Conditioned media of human CRC epithelial cells and CRC myofibroblasts induced anchorage-independent growth. a Percent efficiency of anchorage independent growth in soft agar assays of murine NIH3T3 cells that overexpressed GFP or KRAS12V by lentiviral transduction, and SW48 and SW620 cells. b Increased anchorage independent growth of malignant NIH3T3-KRAS12V cells with addition of conditioned-media from SW620 cells that were cultured under stressed serum-free versus normal serum containing media or control growth media alone. c Serum-free and serum containing conditioned media from SW620 and CRC MF cells maximally induced anchorage independent growth of non-transformed NIH3T3 cells, respectively. d Relative cytokines levels in SW620 and CRC-MF condition media in the presence and absence of serum. e Secreted CXCL1 and IL8 levels in SW620 and CRC-MF conditioned media in the presence and absence of serum as measured by ELISA. Representative experiments were shown from n ≥3 experiments. P values were as indicated: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
Overexpression of CXCL1 and IL8 mRNA levels in human CRC development. a, b Box plots of CXCL1 (a) and IL8 (b) mRNA expressions in human mucosal tissues of normal colon (NC), normal liver (NL), colon adenoma (A), stages I-IV primary colorectal adenocarcinoma (I-IV), and CRC metastases to liver (Li) and lung (Lu). c Box plots of CXCL1 mRNA expressions in human primary CRC stratified according to TNM stages. d Box plots of IL8 mRNA expressions in human primary CRC stratified according to TNM stages. e Percentages of human colorectal malignant tissues with elevated CXCL1 mRNA expression occurred early in most human adenoma (A) and decreased slightly with cancer metastasis. f Percentages of human colorectal malignant tissues with elevated IL8 mRNA expression occurred predominantly at time of conversion from adenoma to stage I–II CRC and remained elevated with disease progression. “−” and “+” indicated normal mRNA levels and those that were three standard deviations above normal, respectively. SI represented arbitrary unit of signal intensity. Patient numbers (n) were as indicated.
Figure 3
Figure 3
Highly elevated levels of CXCL1, but not IL8, associated with poor prognosis in stage IV human CRC. a–d Kaplan–Meier estimates of overall survival in stages IV (a, b) and II-III (c, d) CRC patients that were stratified according to normal (gray line; +/−), and upper-quartile (color line; ++) ranges of CXCL1 (a, c) or IL8 (b, d) levels. The patient numbers at risk (n) were as indicated for the groups.
Figure 4
Figure 4
Induction of anchorage-independent growth by SW620 and CRC-MF conditioned media was dependent on CXCL1. a Neutralizing antibodies to CXCL1, in comparison to irrelevant isotype-specific control IgG Ab, inhibited the anchorage independent growth of non-transformed NIH3T3 cells induced by serum-free SW620 and serum-containing CRC-MF conditioned media. b Neutralizing antibodies to CXCL1 inhibited the anchorage independent growth of malignant SW620 cells in comparison to irrelevant isotype-specific control IgG Ab. c, d SW620 cells stably expressing two different CXCL1 specific shRNAs secreted lower CXCL1 levels (c) and had suppressed soft agar growth (d) in comparison to control shRNA. p values were as indicated: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
CXCL1 contributed to in vivo human CRC tumor growth. a SW620 cells stably expressing two distinct CXCL1 specific shRNAs had suppressed xenograft tumor growth in immunocompromised mice (n = 4 per group) with tumor images at right. b Immunofluorescence of human CRC detected CXCL1 protein expression (red) in CRC epithelia (asterisks) and co-localization with tumor myofibroblasts (arrowheads), which expressed α-smooth muscle actin (green). c, d In primary stage I–IV human CRC, mRNA levels of CXCL1 show a moderate correlation with CXCR2 (Spearman r = 0.34; CI 0.20–0.48) but not CXCR1 (Spearman r = −0.04; 95% CI −0.20 to 0.11).
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