Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.     

Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy

Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy.     

Expression of heat shock protein 27 in human renal cell carcinoma

Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.     

Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.     

Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect

Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.     

LINC00961 restrains cancer progression via modulating epithelial–mesenchymal transition in renal cell carcinoma

Recently, long noncoding RNA have been identified as new gene regulators and prognostic biomarkers in various cancers, including renal cell carcinoma (RCC). The expression and biological roles of LINC00961 have been reported in many human cancers. However, up to date, no study of LINC00961 has been shown in RCC. Currently, we aimed to investigate the function of LINC00961 in RCC progression. Interestingly, we observed that LINC00961 could act as a novel biomarker in predicting the diagnosis of RCC. Then, we found that LINC00961 was greatly downregulated in RCC cell lines (Caki-1, Caki-2, 786-O, A498, and ACHN cells) compared with normal renal cell lines (HK-2 cells). Then, 786-O cells and ACHN cells were infected with LV-LINC00961. As displayed in our current study, LINC00961 overexpression could obviously suppress the proliferation and survival of RCC cells in vitro. In addition, RCC cell apoptosis was greatly induced and cell cycle progression was blocked in G1 phase by upregulation of LINC00961 in 786-O cells and ACHN cells. Subsequently, we found that LV-LINC00961 was able to restrain RCC cell migration and cell invasion capacity. Meanwhile, the messenger RNA and protein expression levels of epithelial–mesenchymal transition (EMT)-associated markers Slug and N-cadherin in RCC cell lines were dramatically inhibited by overexpressing LINC00961. Finally, the in vivo experiment was carried out and we observed that LINC00961 could inhibit RCC development through modulating EMT process. Taken these together, it was indicated in our study that LINC00961 was involved in RCC progression through targeting EMT pathway.     

Tissue transglutaminase expression is necessary for adhesion,metastatic potential and cancer stemness of renal cell carcinoma

Tissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family and shown to play a critical role in the development and progression of drug resistance malignancies. We have previously showed the association of TG2 upregulation with progression and metastasis of renal cell carcinoma (RCC) and low disease-free survival. In the present study we further investigate the role of TG2 in cell adhesion, migration and invasion of RCC by silencing TG2 expression in Caki-2 and A-498 primary site and Caki-1 and ACHN metastatic site RCC cell lines. Downregulation of TG2 expression led up to a 60% decrease in actin stress fiber formation and adhesion to β 1 integrin (ITGB1) substrates fibronectin, collagen type I and laminin in both primary and metastatic site RCC cell lines. In addition, treatment with siRNAs against TG2 impaired the migration capacity and cellular invasiveness of ITGB1 substrates in all 4 RCC cell lines. Lastly, the knockdown of TG2 in metastatic Caki-1 cells diminished the expression of CD44, CD73-and CD105 cancer stem cell-like markers. We conclude, for the first time, that TG2 expression is critical for cancer cell adhesion, migration, invasiveness and cancer cell-stemness during RCC progression and dissemination. Therefore, combined targeting of TG2 with drugs widely used in the treatment of RCC may be a promising therapeutic strategy for RCC.     

Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.     

Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma

Heat shock proteins (HSP) are families of highly conserved proteins which are induced in cells and tissues upon exposure to extreme conditions causing acute or chronic stress. They exhibit distinct functions and have been implicated in the pathogenesis of a number of diseases, including cancer. A causal relationship between HSP expression and immunogenicity has been demonstrated in murine and human tumors and is also associated with the immune response. In order to investigate the correlation of HSP expression and their immunogenic potential in renal cell carcinoma (RCC), we here analyzed (i) the protein expression profile of various members of the HSP family in untreated and interferon (IFN)-gamma treated RCC cell lines as well as normal kidney epithelium, and (ii) the anti-heat shock protein reactivity in sera derived from RCC patients and healthy controls using proteomics-based techniques. A heterogeneous expression pattern of members of the HSP families was demonstrated in RCC cell lines and in cells representing normal renal epithelium. In some cases the expression rate is moderately altered by IFN-gamma treatment. In addition, a distinct anti-heat shock protein reactivity could be detected in autologous and allogeneic sera from RCC patients and healthy controls. These data suggest that HSP play a role in the immunogenicity of RCC and thus might be used for the design of immunization strategies to induce a potent antitumor response in this disease.     

Multiple biomarker tissue arrays: A computational approach to identifying protein-protein interactions in the EGFR/ERK signalling pathway

ABSTRACT: BACKGROUND: Many studies have demonstrated genetic and environmental factors that lead to renal cell carcinoma (RCC) and that occur during a protracted period of tumourigenesis. It appears suitable to identify and characterise potential molecular markers that appear during tumourigenesis and that might provide rapid and effective possibilities for the early detection of RCC. EGFR activation induces cell cycle progression, inhibition of apoptosis and angiogenesis, promotion of invasion/metastasis, and other tumour promoting activities. Over-expression of EGFR is thought to play an important role in tumour initiation and progression of RCC because up-regulation of EGFR has been associated with high grade cancers and a worse prognosis. METHODS: Characterisation of the protein profile interacting with EGFR was performed using the following: an immunohistochemical (IHC) study of EGFR, a comprehensive computational study of EGFR protein-protein interactions, an analysis correlating the expression levels of EGFR with other significant markers in the tumourigenicity of RCC, and finally, an analysis of the utility of EGFR for prognosis in a cohort of patients with renal cell carcinoma. RESULTS: The cases that showed a higher level of this protein fell within the clear cell histological subtype (p = 0.001). The EGFR significance statistic was found with respect to a worse prognosis. In vivo significant correlations were found with PDGFR-beta, Flk-1, Hif1-alpha, proteins related to differentiation (such as DLL3 and DLL4 ligands), and certain metabolic proteins such as Glut5. In silico significant associations gave us a panel of 32 EGFR-interacting proteins (EIP) using the APID and STRING databases. CONCLUSIONS: This work summarises the multifaceted role of EGFR in the pathology of RCC, and it identifies EIPs that could help to provide mechanistic explanations for the different behaviours observed in tumours.     

Candidate biomarkers in renal cell carcinoma

Although the human genome has been decoded, the knowledge about the pathogenesis of diseases including cancer is still limited. By focusing on renal cell carcinoma (RCC) we here summarize the data of various research groups analyzing the protein/peptide expression profiles of tumor lesions/cell lines or serum obtained from patients and respective controls. Different powerful approaches such as 2-DE, PROTEOMEX/SERPA/SPEARS, and T cell epitope discovery upon elution of MHC class I-bound peptides in combination with MS/LC-MS/MS revealed 500 differentially expressed proteins. The overlap in target recognition limits the pool to 299 unique protein identities, but only few thereof (12%) have been validated. The management, analysis, and interpretation of the distinct data sets derived from 27 publications required bioinformatic restructuring of the results. However, the comprehensive analysis of the results expands the knowledge about the pathophysiology of RCC in particular of the most prominent clear cell subtype by providing information on the differentially expressed proteins, their regulation status in RCC compared to normal kidney epithelium next to additional information on MHC-presented T cell epitopes and on serological targets. Despite the low number of validated differentially expressed proteins some of them might serve as candidate biomarkers for the diagnosis and/or as therapeutic targets.     

Regulation of chondrocyte differentiation by actin-severing protein adseverin

The importance of actin organization in controlling the chondrocyte phenotype is well established, but little is known about the cytoskeletal components regulating chondrocyte differentiation. Previously, we have observed up-regulation of an actin-binding gelsolin-like protein in hypertrophic chondrocytes. We have now identified it as adseverin (scinderin). Adseverin is drastically up-regulated during chondrocyte maturation, as shown by Northern blot analysis, in situ hybridization, and real-time RT-PCR. Its expression is positively regulated by PKC and MEK signaling as shown by inhibitory analyses. Over-expression of adseverin in non-hypertrophic chondrocytes causes rearrangement of the actin cytoskeleton, a change in cell morphology, a dramatic (3.5-fold) increase in cell volume, and up-regulation of Indian hedgehog (Ihh) and of collagen type X--all indicative of chondrocyte differentiation. These changes are mediated by ERK1/2 and p38 kinase pathways. Thus, adseverin-induced rearrangements of the actin cytoskeleton may mediate the PKC-dependent activation of p38 and Erk1/2 signaling pathways necessary for chondrocyte hypertrophy, as evidenced by changes in cell morphology, increase in cell size and expression of the chondrocyte maturation markers. These results demonstrate that interdependence of cytoskeletal organization and chondrogenic gene expression is regulated, at least in part, by actin-binding proteins such as adseverin.     

Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.     

Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.     

Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.     

The conversion of mouse skin squamous cell carcinomas to spindle cell carcinomas is a recessive event

Squamous carcinomas of both human and rodent origin can undergo a transition to a more invasive, metastatic phenotype involving reorganization of the cytoskeleton, loss of cell adhesion molecules such as E-cadherin and acquisition of a fibroblastoid or spindle cell morphology. We have developed a series of cell lines from mouse skin tumors which represent different stages of carcinogenesis, including benign papillomas, and clonally related squamous and spindle carcinomas derived from the same primary tumor. Some spindle cells continue to express keratins, but with a poorly organized keratin filament network, whereas in others no keratin expression is detectable. All of the spindle cells lack expression of the cell adhesion molecule E-cadherin and the desmosomal component desmoplakin. Loss of these cell surface proteins therefore appears to precede the destabilization of the keratin network. At the genetic level, it is not known whether such changes involve activation of dominantly acting oncogenes or loss of a suppressor function which controls epithelial differentiation. To examine this question, we have carried out a series of fusion experiments between a highly malignant mouse skin spindle cell carcinoma and cell lines derived from premalignant or malignant mouse skin tumors, including both squamous and spindle carcinoma variants. The results show that the spindle cell phenotype as determined by cell morphology and lack of expression of keratin, E-cadherin, and desmoplakin proteins, is recessive in all hybrids with squamous cells. The hybrids expressed all of these differentiation markers, and showed suppression of tumorigenicity to a variable level dependent upon the tumorigenic properties of the less malignant fusion partner. Our results suggest that acquisition of the spindle cell phenotype involves functional loss of a gene(s) which controls epithelial differentiation.