PTHrP increases xenograft growth and promotes integrin alpha6beta4 expression and Akt activation in colon cancer

Abstract

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Expression of PTHrP and phosphatidylinositol 3-kinase (PI3-K) pathway components correlates with the severity of colon carcinoma. Here we observed a positive effect of endogenous PTHrP on LoVo (human colon cancer) cell proliferation, migration, invasion, integrin alpha6 and beta4 expression, and p-Akt levels. There was a direct correlation between PTHrP expression and anchorage-independent cell growth. PTHrP significantly increased xenograft growth; tumors from PTHrP-overexpressing cells showed increased expression of integrins alpha6 and beta4, and PI3-K pathway components. The higher expression of PTHrP in human colon cancer adenocarcinoma vs. normal colonic mucosa was accompanied by increased integrin alpha6 and beta4 levels. Elevated PTHrP expression in colon cancer may thus upregulate integrin alpha6beta4 expression, with consequent PI3-K activation. Targeting PTHrP might result in effective inhibition of tumor growth, migration, and invasion.

Figure 1. Expression of the integrin α6 and β4 subunits and p-Akt in LoVo cells with suppressed PTHrP expression

(A) mRNA levels of PTHrP and the integrin α6 and β4 subunits were measured by reverse transcription/real-time PCR. (B) PTHrP secretion was measured using an immunoradiometric assay. (C and D) Cells were stained with phycoerythrin-conjugated antibodies against the integrin 6 or 4 subunits, and analyzed by FACS. (E) Cells were stained with an Alexa Fluor 488-conjugated anti-p-Akt antibody and analyzed by FACS. In (C-E), positive cells are those whose log fluorescence intensity (MFI) is greater than that of the isotype control antibody-stained cells. Values are expressed relative to the control (scrambled siRNA) value, set at 100%. sc = scrambled siRNA control; si1 and si2 = two independent cell lines derived by transfection with independent PTHrP siRNAs. In (A-E), each bar is the mean ± SEM of three independent experiments. * = Significantly different from the control value (P < 0.001).

Figure 2. Effect of endogenous PTHrP on LoVo cell proliferation, migration and invasion

(A) Proliferation. Cells were plated in 96-well dishes at 10⁴ cells/well. Cell number was determined at the indicated time intervals. For si and sc, each point is the mean ± SEM of three independent experiments each for si1 and si2 or the respective scrambled (sc) siRNA control. (B and C) Migration and invasion. Cells (0.5 × 10⁶) were loaded with Calcein-AM and plated onto FluoroBlok inserts in the absence of FBS. To measure invasion, the inserts were overlaid with Matrigel. FBS (10%) was used as the chemoattractant. Migration and invasion were measured after 4 h. In (B and C), 1 and 2 refer to two independent PTHrP siRNA-transfected cell lines. Each bar is the mean ± SEM of three independent experiments. (D) Migration was analyzed using a monolayer scratch assay. Images at 0 and 5 days after wounding are shown. The arrowed lines mark the edges of the monolayer. Magnification, ×10. (E) Quantitation of cell migration 3, 4, and 5 days after wounding the cell monolayer. si is the mean ± SEM of three independent experiments each for si1 and si2. * = Significantly different from the control value (P < 0.001).

Figure 3. Effects of PTHrP on anchorage-independent cell growth

Assays to determine colony formation in soft agar were performed in 60 mm dishes containing a bottom layer consisting of 1.5 ml culture medium containing 0.4% (w/v) agar. Cells (1 × 10⁴) were plated in a top layer of 0.3% agar. (A) After 2 weeks in culture, the plates were photographed at 40 × magnification, and clone size was measured using the ImageJ software (NIH). (B) Photographs were also taken at 10 × magnification to measure clone frequency. All clones in focus > 50 μm in size were measured. (C) Colony size. Each bar is the mean ± SEM of 20 colonies for each of three independent clones overexpressing PTHrP (+), three independent empty vector-transfected clones (V), two independent cell lines (si1 and si2) generated by transfection with independent PTHrP siRNAs (si), or the respective scrambled siRNA sequences (sc), or parental cells (P). (D) Average number of colonies. Each bar is the mean ± SEM of three fields per plate for each of the cell lines described in (C). (C and D) * = Significantly different from the control value (P < 0.001).

Figure 4. Effects of PTHrP on LoVo cell xenograft growth in nude mice

Athymic nude mice were injected with LoVo cells overexpressing PTHrP, or with control cells. (A) Representative tumors from four independent PTHrP-overexpressing clones [+ (1) to + (4)], three independent empty vector-transfected clones (V1 to V3), and parental cells (P). (B) Mean volumes of tumors produced by PTHrP-overexpressing cells (○), empty vector transfectants (▼), and parental cells (●). (C) Tumor weight at time of sacrifice. In (B) and (C), each point or bar is the mean ± SEM of tumor volumes (B) or weight (C) from four independent PTHrP-overexpressing clones, three independent empty vector clones and parental cells (6 animals/clone).

Figure 5. Immunohistochemical analysis of xenografts from PTHrP-overexpressing and control LoVo cells

(A) Staining for PTHrP, integrin α6, integrin β4, and components of the PI3-K pathway is shown. H&E = sections stained with haematoxylin and eosin. IgG = sections stained with an anti-rabbit IgG antibody (negative control). + = xenograft sections from PTHrP-overexpressing cells; V = xenograft sections from control (empty-vector-transfected) cells. Each panel is representative of five sections for each of six tumors from mice injected with one of four independent PTHrP-overexpressing clones or three empty vector-transfected clones. Magnification, × 40. The staining intensity for each protein was graded between 0–3, and was obtained by comparing the level of staining present and the proportion of stained cells in sections from PTHrP-overexpressing cells vs. that of control cells. (B) The pie charts summarize the expression patterns obtained from 120 sections for PTHrP-overexpressing cells (5 slides for each of 4 independent clones; 6 animals/clone) and 90 sections for the empty vector transfectants (5 slides for each of 3 independent clones; 6 animals/clone).

Figure 6. Immunohistochemical analysis for PTHrP and the integrin α6 and β4 subunits in human colorectal cancer and patient-matched normal mucosa

(A) Representative expression patterns are shown for tubulovillous adenoma (Stage 0) and colorectal carcinoma (Stage II), each with patient-matched normal mucosa. Each panel is representative of a minimum of five slides for each of 20 patients. Magnification, × 40. The staining intensity was graded between 0 and 3, as described in Fig. 5. (B) The pie charts summarize the expression patterns obtained from sections from 20 patients (5 slides/patient). The chart for normal sections summarizes two sets of data (normal control data from Stage 0 and Stage II patients).