Rapid development of salivary gland carcinomas upon conditional expression of K-ras driven by the cytokeratin 5 promoter

Abstract

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of DeltaN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis.

Figure 1

A: Gross anatomy of the salivary glands in a K5-tet-on/tet-o-ras animal. All glands are grossly distorted and enlarged showing a multinodular surface (SM: submaxillary, SL: sublingual). The accessory buccal glands (Acc) are also enlarged. The inset shows the glands already visible as tumor-like structures protruding through the neck skin. The abnormal gross anatomy is better depicted in the dissected gland (B) surface (SM: submaxillary, SL: sublingual, P: parotid); C: Relatively flat end even surface of normal salivary glands for comparison. D–G: Normal histology of the accessory salivary glands. These are microscopic mixed, mucous, and serous glands (inset, ductal structure), inconspicuous unless enlarged, located beneath the superficial muscular layer close to the buccal mucosa. D: Location of the glands in relation to the oral mucosa and the skin (Alcian Blue, 64×). E: Serous (white arrow) and mucinous (black arrow) lobules are separated at this level by a thin connective wall. In deeper sections both components coalesce (F), and the serous component disappears in even deeper sections (G). Excretory ducts are indicated by an arrowhead. (Different amplifications of pictures taken at 10× are shown.)

Figure 2

Dysplastic changes with solid squamous metaplastic proliferation (A) and adenosis (B) are evident in the submaxillary gland. The parenchyma of the sublingual gland seems unaffected, but the extralobular ducts show focal intraductal cribiform proliferation (inset). Magnifications: ×40 and ×100 (inset).

Figure 3

Advanced dysplastic changes with fibroadenoma-like changes (A and inset, upper right corner) and complex adenosis (B and inset, lower right corner). Few mononuclear cells can be seen in the stroma. These are a later development in the evolution of the process; no infiltrates are seen in early time points. Magnifications: ×40 and ×100 (insets).

Figure 4

A: This multifocal squamous cell carcinoma arises in an otherwise unaffected submaxillary gland; no signs of hyperplasia or dysplasia are observed. The inset shows a distinct limit between neoplasia and normal tissue (arrow). B: Multifocal spindle-cell carcinomas in a sublingual gland arising from ductal structures; the inset depicts the border between the carcinoma and the normal tissue; a fibrous pseudocapsule with inflammatory mononuclear cells is seen (arrow). C: Moderately differentiated multifocal squamous cell carcinoma arising in a submaxillary gland; the tumors form solid masses that infiltrate the surrounding stroma. Distorted mucous acini are seen in the periphery. D: Infiltrating spindle-shaped carcinoma, with a desmoplastic stroma. E: Early carcinoma arising in a dysplastic sublingual gland. Note that, although the lesion is small, all of the cells display malignant features; a rapid transition to invasion characterized these tumors, with no evident dysplasias. F: Metastatic squamous cell carcinoma in a cervical lymph node; the arrows indicate remnants of the lymph node parenchyma. Magnifications: ×20 (A); ×30 (B); ×45 (C–F); and ×100 (insets).

Figure 5

Panoramic view of a buccal accessory salivary gland with a wide spectrum of histological changes from hyperplasia to carcinoma, showing its relations with the oral cavity. Note that, unlike the extraorbital lacrimal glands, these structures contain mucous acini, remnants of which are shown. A squamous papilloma of the oral epithelium that has an area of invasive carcinoma can also be seen. Magnification: ×40.

Figure 6

A: β-Galactosidase staining; isolated ductal cells are positive. B: Immunostaining for keratin 5 in sublingual (upper figure) and submaxillary (lower figure) glands reveals positive staining in most ductal cells as well as in myoepithelial cells. C: Higher magnification of K5 expression in the sublingual gland. D: Submaxillary gland immunoreacted with K5. E: K5-tet-on/tet-o-ras mice. K5 is expressed in all proliferating cells in a hyperplastic excretory duct. Magnifications: ×40 (B); ×60 (C–E); and ×120 (A).

Figure 7

A and B: K5 staining in the hyperplastic area of the submaxillary and sublingual glands, respectively, showing strong immunoreactivity in the proliferating cells. C: Strong K5 immunoreactivity in carcinomatous cells (arrow), as well as in a lymph node metastasis (inset). D: Distribution of smooth muscle actin staining in a dysplasia, with scattered cells surrounding glandular structures. In E, the carcinomatous area on the left side of the picture is negative (arrows), whereas the hyperplastic glands on the right side show positive periglandular myoepithelial cells. F and G: Scattered immunoreactivity for p63 in normal submaxillary and sublingual glands respectively (empty arrows: ductal cells; arrows: myoepithelial cells). In H, a higher number of p63-immunoreactive cells are seen in the carcinomatous (Ca) as compared with a hyperplastic area (Hyp). In both cases the proportion of positive cells is higher than that of normal tissues. I and J: a higher proportion of BrdU-retaining cells in hyperplastic areas (empty arrows) as compared with normal areas (arrows) in submaxillary and sublingual glands, respectively. Magnifications: ×30 (A–C); ×40 (D, E, H, I, and J); and ×60 (F and G).