Autocrine CSF-1 and CSF-1 receptor coexpression promotes renal cell carcinoma growth

Abstract

Renal cell carcinoma is increasing in incidence but the molecular mechanisms regulating its growth remain elusive. Coexpression of the monocytic growth factor colony-stimulating factor (CSF)-1 and its receptor CSF-1R on renal tubular epithelial cells (TEC) will promote proliferation and antiapoptosis during regeneration of renal tubules. Here, we show that a CSF-1-dependent autocrine pathway is also responsible for the growth of renal cell carcinoma (RCC). CSF-1 and CSF-1R were coexpressed in RCCs and TECs proximally adjacent to RCCs. CSF-1 engagement of CSF-1R promoted RCC survival and proliferation and reduced apoptosis, in support of the likelihood that CSF-1R effector signals mediate RCC growth. In vivo CSF-1R blockade using a CSF-1R tyrosine kinase inhibitor decreased RCC proliferation and macrophage infiltration in a manner associated with a dramatic reduction in tumor mass. Further mechanistic investigations linked CSF-1 and epidermal growth factor signaling in RCCs. Taken together, our results suggest that budding RCC stimulates the proximal adjacent microenvironment in the kidney to release mediators of CSF-1, CSF-1R, and epidermal growth factor expression in RCCs. Furthermore, our findings imply that targeting CSF-1/CSF-1R signaling may be therapeutically effective in RCCs.

Figure 1. CSF-1R and CSF-1 are expressed in RCC, but not PC

We detected robust CSF-1R and CSF-1 expression in RCC and TEC adjacent to RCC as compared with areas distant to RCC and normal human kidney. We did not detect a rise in PC. CSF-1R and CSF-1 Ab specificity was verified u s i n g p e p t i d e p r e-absorption. Representative photomicrographs (magnification 20×) are shown. B. CSF-1R expression in TEC correlates with CSF-1 expression in RCC and TEC adjacent to RCC, but not distant to RCC. Values are the mean ± SEM.

Figure 2. CSF-1R and CSF-1 expression on RCC and TEC that are adjacent to RCC correlate with infiltrating Mø, but not CD3⁺ T cells

A. We evaluated the presence of CD68⁺ and CD3⁺ leukocytes in formalin-fixed sections by immunostaining. We confirmed staining specificity using isotype control Abs. Representative photomicrographs are shown (magnification 20×, enlargement 40×). B. Correlations of CSF-1R and CSF-1 expression with CD68⁺ and CD3⁺ leukocytes in RCC and the TEC in areas adjacent and distant to RCC. Values are the mean ± SEM.

Figure 3. CSF-1 and CSF-1R co-expression on RCC promotes survival/proliferation, thereby leading to increased RCC growth

Using serial sections of kidney biopsy specimens from patients with RCC, we probed for CSF-1 and CSF-1R expression, CSF-1R phosphorylation at Y723 and proliferation in tubules. CSF-1R and CSF-1 are co-expressed on TEC and CSF-1R and tyrosine phosphorylated CSF-1R are co-expressed on TEC (immunostaining). Note, proliferating Ki67⁺ TEC in tubules co-expressing CSF-1 and CSF-1R. CSF-1R and CSF-1 Ab specificity was verified using peptide pre-absorption, Ki67 and phospho-Y723 CSF-1R Ab specificity was verified using rabbit IgG (right panels). Representative photomicrographs and correlation graphs. Magnification 20×, enlargement 40×. Values are means ± SEM; n=8–9/group.

Figure 4. CSF-1 and CSF-1R are increased on TEC by mediators released from RCC and, to a lesser degree, Mø

Top panel: CSF-1 expression of cultured TEC (HK2 cell line) following incubation with supernatant of stimulated RCC (786-0 cell line) or Mø (HL60 cell line). Representative data is shown for stimulation with TNF-α, stimulations with TNF-α/LPS and Phorbol 12-Myristate 13 Acetate (TPA) showed comparable results (data not shown). Supernatants were added to the cultured TEC cell line (HK2) undiluted, diluted 1:1, 1:4 and 1:8 with media from stimulated RCC cells (786-0) or Mø (HL60). Bottom panel: CSF-1 and CSF-1R transcript expression of cultured TEC (HK2 cell line) following incubation with supernatant of stimulated RCC or Mø. Results are representative of 3 separate experiments. Values are means ± SEM.

Figure 5. CSF-1R and CSF-1 are expressed on RCC lines

A. Left panel: CSF-1R transcript expression in RCC cells (786-0) determined by real-time PCR. Normal human TEC (HK2) human breast carcinoma line, (MCF-7) and the human leukemic cell line (HL60) stimulated with TPA served as positive controls. TPA stimulation of the human T cell line (JURKAT) served as a negative control. Results are representative of 3 separate experiments; means ± SEM. Right panel: RCC lines (786-0), HK2 cell line and MCF-7 lines stimulated with TPA and stained with anti-CSF-1R and control rabbit IgG. Results are representative of 3 separate experiments; means ± SEM. B. Left panel: CSF-1 transcript expression in RCC cells determined by real-time PCR. HK2, MCF-7 lines stimulated with TNF-α/LPS served as positive controls. Results are representative of 3 separate experiments; means ± SEM. Right panel: Stimulation of RCC cells increases secretion of CSF-1 protein. Following exposure of RCC cells to increasing concentrations of TNF-α/LPS for 48h, we analyzed CSF-1 in the supernatant by ELISA. Results are representative of 3 separate experiments. Values are means ± SEM.

Figure 6. CSF-1 engaging with CSF-1R on RCC promotes survival/proliferation, and suppresses RCC apoptosis thereby leading to increased RCC growth

A. CSF-1 stimulates survival/proliferation (MTT assay) of RCC (786-0) and blocking with anti-CSF-1R Ab suppresses CSF-1 mediated RCC proliferation. HK2 and MCF-7 lines served as positive controls. Results are representative of 4 separate experiments; means ± SEM. B. RCC were incubated with TNF-α/LPS in the presence and absence of anti-CSF-1R Ab for 72h prior to determination of cell mass by MTT assay. HK2 and MCF-7 lines served as positive controls. Results are representative of 3 separate experiments. Values are means ± SEM. C. TNF-α /LPS stimulated RCC are self-protective through a CSF-1-dependent mechanism that dampens RCC apoptosis. RCC were cultured for 72h in the absence of CSF-1. Blocking the CSF-1R increases TNF-α/LPS-induced TEC apoptosis. Means ± SEM; n=6–8/group.

Figure 7. Blocking CSF-1R signaling in vivo inhibits RCC growth

A. CSF-1 stimulates proliferation (MTT assay) of RCC (786-0) and blocking with CSF-1R tyrosine kinase inhibitor suppresses CSF-1 mediated RCC proliferation. HK2 line served as a positive control. Results are representative of 2 separate experiments; means ± SEM. B. RCC (786-0) and HK2 cells were implanted under the renal capsule of athymic nu/nu Balb/c mice. CSF-1R tyrosine kinase inhibitor was injected beginning 1 day after surgery for 21 days. Mice were sacrificed 22 days following surgery. Growth (mass), proliferation (Ki67) and infiltration of CD68⁺ leukocytes in RCC was suppressed in mice treated with the CSF-1R tyrosine kinase inhibitor compared with PBS treated controls. Proliferating cells and CD68⁺ leukocytes are circled. Representative photomicrographs (T= tumor). Values are means ± SEM; n=4–5/group.

Figure 8. EGF mediates CSF-1-dependent enhanced proliferation and suppressed apoptosis of RCC

A. EGF stimulates RCC cells to express CSF-1. CSF-1 measured in the supernatant of RCC cells (786-0 and Caki) by ELISA. Results are representative of 3 separate experiments. Values are means ± SEM. B. EGF stimulates proliferation of RCC cells and anti-EGFR Ab or anti CSF-1R Ab suppresses EGF dependent proliferation of RCC cells. HK2 cell line served as a positive control. Proliferation assessed by the MTT assay. Values are means ± SEM. Results are representative of 4 separate experiments. C. EGF mediated CSF-1 expression suppresses RCC apoptosis. RCC were cultured for 72h in the absence of CSF-1. Cells were stimulated with concentrations of TNF-α and LPS. EGF stimulation dampens apoptosis, and blocking the CSF-1R with anti-CSF-1 Ab or blocking the EGFR with anti-EGFR Ab increases TNF-α/LPS-induced TEC apoptosis. Means ± SEM; n=6/group. D. RCC and Mø release mediators that stimulate TEC to express EGF. RCC (786-0) and Mø were stimulated with TNF-α. Supernatants were added to the cultured TEC cell line (HK2) undiluted, diluted 1:1, 1:4 and 1:8 with media from stimulated RCC cells (786-0) or Mø (HL60). EGF expression was measured by ELISA. Note, comparable results stimulating RCC and Mø with TNF-α/LPS and TPA (data not shown).