Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma

Abstract

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Figure 1.

Induction of β3 expression by β3/Ad5. A: Cell surface expression of virally transduced β3 integrin subunit. Virus-infected melanoma cells were trypsinized, sequentially incubated with anti-β3 MAb (SSA6) and FITC-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. The x axis indicates relative fluorescence intensity (log units); the y axis shows the relative cell number. - - - and ——, lacZ/Ad5- and β3/Ad5-infected cells, respectively. B: Complex formation of virus-induced β3 with endogenous αv. Cell lysates of infected melanoma cells were prepared after metabolic labeling, normalized for radioactivity, immunoprecipitated with β3-specific SSA6 MAb, and subjected to electrophoresis. Gels were fixed, dried, and exposed to x-ray film.

Figure 2.

In vitro growth of melanoma cells after β3 overexpression. Two days after viral infection, 2 × 10⁵ cells were seeded into six-well tissue culture plates. Cell growth was monitored on days 1, 4, and 7 using a Coulter counter. Average cell number from triplicate wells was plotted for WM1552 and SBcl2 cells.

Figure 3.

Cell growth in soft agar of melanoma cells overexpressing β3. Melanoma cells were infected at a multiplicity of infection of 20 and, 2 days later, resuspended in 0.25% agar, seeded on an acellular layer, and fed regularly. After 3 to 4 weeks of culture, colony-forming efficiency was determined as the percentage of cells forming colonies containing four or more cells. Ten randomly chosen high-power fields were examined for each condition. *Statistical significance using Student’s t-tests; P values were 0.009 and 0.007 for SBcl2 and WM1552C cells, respectively.

Figure 4.

Effect of β3 overexpression on melanoma invasion and survival in three-dimensional skin reconstructs. Virus-infected SBcl2 melanoma cells were incorporated into the epidermis of skin reconstructs as described in Materials and Methods. Mature reconstructs were harvested, fixed, and embedded in paraffin. β3/Ad5-infected SBcl2 cells grew in an invasive pattern reminiscent of VGP primary melanoma (A and C), whereas lacZ/Ad5-infected cells spread horizontally, resembling RGP primary melanoma (B and D). Control virus-infected cells at the dermal/epidermal junction displayed apoptotic features, including nucleus condensation, membrane blebbing, and presence of apoptotic bodies. β3/Ad5-infected cells were completely negative for staining by the ApopTag in situ apoptosis detection kit (E), whereas lacZ/Ad5-infected cells stained positive (F). Magnification, ×100 (A, B, E, and F) and ×259 (C and D).

Figure 5.

Tumorigenicity of melanoma cells overexpressing β3 subunit. Ad5-infected cells were trypsinized and resuspended in growth medium. SCID mice were injected subcutaneously with 3 × 10 cells/mouse in a volume of 100 μl (five mice/group), and tumor size was determined at day 7. Student’s t-tests confirmed statistically significant differences between groups with P values <0.05 (SBcl2, P = 0.006; WM1552C, P = 0.001).