Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis.

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.

Does Molecular Profiling of KRAS-Mutant Non-Squamous Non-Small Cell Lung Cancer (NSCLC) Help in Treatment Strategy Planning?

Background: Several studies suggest that patients with KRAS-mutant NSCLC fail to benefit from standard systemic therapies and do not respond to EGFR inhibitors. Most recently, KRAS 12c data suggest specific treatment for improving ORR and OS. There is a clear need for therapies specifically developed for these patients. Moreover, data that might be suggestive of a response to specific therapies, such as BRCA1, are needed, and two mutations that were studied in other malignancies show more response to PARP inhibitors. Molecular profiling has the potential to identify other potential targets that may provide better treatment and novel targeted therapy for KRAS-mutated NSCLC. Methods: We purified RNA from archived tissues of patients with stage I and II NSCLC with wild-type (wt) and mutant (mt) KRAS tumors; paired normal tissue adjacent to the tumor from 20 and 17 patients, respectively, and assessed, using real-time reverse transcriptase−polymerase chain reaction (RT-PCR), the expression of four genes involved in DNA synthesis and repair, including thymidylate synthase (TS), BRCA1, ECCR1, RAP80, and the proto-oncogene SRC. Additionally, we assessed the expression of PD-L1 in mt KRAS tumors with immunohistochemistry using an antibody against PD-L1. Results: Our results show that in mtKRAS tumors, the level of expression of ERCC1, TS, and SRC was significantly increased in comparison to paired normal lung tissue (p ≤ 0.04). The expression of BRCA1 and RAP80 was similar in both mt KRAS tumors and paired normal tissue. Furthermore, the expression of BRCA1, TS, and SRC was significantly increased in wt KRAS tumors relative to their expression in the normal lung tissue (p < 0.044). The expression of ERCC1 and RAP80 was similar in wt KRAS tumors and paired normal tissue. Interestingly, SRC expression in mtKRAS tumors was decreased in comparison to wt KRAS tumors. Notably, there was an expression of PD-L1 in the tumor and stromal cells in a few (5 out of 20) mtKRAS tumors. Our results suggest that a greater ERCC1 expression in mt KRAS tumors might increase platinum resistance in this group of patients, whereas the greater expression of BRCA1 in wt KRAS tumor might be suggestive of the sensitivity of taxanes. Our data also suggest that the combination of an SRC inhibitor with a TS inhibitor, such as pemetrexed, might improve the outcome of patients with NSCLC and in particular, patients with wt KRAS tumors. PD-L1 expression in tumors, and especially stromal cells, suggests a better outcome. Conclusion: mt KRAS NSCLC patients might benefit from a treatment strategy that targets KRAS in combination with therapeutic agents based on pharmacogenomic markers, such as SRC and BRCA1. mtKRAS tumors are likely to be platinum-, taxane-, and pemetrexed-resistant, as well as having a low level of PD-L1 expression; thus, they are less likely to receive single-agent immunotherapy, such as pembrolizumab, as the first-line therapy. wt KRAS tumors with BRCA1 positivity tend to be sensitive to taxane therapy and, potentially, platinum. Our results suggest the need to develop targeted therapies for KRAS-mutant NSCLC or combine the targeting of oncogenic KRAS in addition to other therapeutic agents specific to the molecular profile of the tumor.

A Structured Program Maximizes Benefit of Lung Cancer Screening in an Area of Endemic Histoplasmosis.

BACKGROUND: Lung cancer screening with low-dose computed tomography has demonstrated at least a 20% decrease in lung cancer-specific mortality, but it has the potential harm of unnecessary invasive procedures performed because of false-positive results. This study reports the outcomes of a structured multidisciplinary lung cancer screening program in an area of endemic histoplasmosis. METHODS: A retrospective review of patients undergoing lung cancer screening from December 2012 to March 2019 was conducted. Findings suggestive of lung cancer were presented to a multidisciplinary thoracic tumor board. Patients were assigned to interval imaging follow-up, additional diagnostic imaging, or referral for an invasive procedure. Invasive procedures were then compared between benign and malignant diseases. RESULTS: A total of 4087 scans were performed on 2129 patients; 372 (9.1%) were suspicious and were presented to a multidisciplinary thoracic tumor board. Ultimately, 108 procedures were performed: 55 bronchoscopies, 7 percutaneous biopsies, and 46 operations. A total of 25 patients (1.2%) underwent bronchoscopy resulting in benign pathologic findings, significantly associated with an indication of an endobronchial lesion (P = .01). All percutaneous biopsy specimens revealed malignancy. Five patients (0.2%) who underwent resection had benign disease. Lung cancer was diagnosed in 67 patients (3.1% of the entire cohort), 46 of whom had stage I or II disease. CONCLUSIONS: Lung cancer screening in a structured, multidisciplinary program successfully identifies patients with early-stage lung cancer with limited unnecessary surgical interventions. Patients with isolated endobronchial lesions should undergo short interval imaging follow-up to avoid bronchoscopy for benign disease. Future studies to minimize unnecessary procedures could incorporate biomarkers and advanced imaging analysis into risk assessment models.

The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis.

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK-/-). Mammary glands from RonTK-/- mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK-/- epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK-/- glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.

Prostate Epithelial RON Signaling Promotes M2 Macrophage Activation to Drive Prostate Tumor Growth and Progression.

Effective treatment of advanced prostate cancer persists as a significant clinical need as only 30% of patients with distant disease survive to 5 years after diagnosis. Targeting signaling and tumor cell-immune cell interactions in the tumor microenvironment has led to the development of powerful immunotherapeutic agents, however, the prostate tumor milieu remains impermeable to these strategies highlighting the need for novel therapeutic targets. In this study, we provide compelling evidence to support the role of the RON receptor tyrosine kinase as a major regulator of macrophages in the prostate tumor microenvironment. We show that loss of RON selectively in prostate epithelial cells leads to significantly reduced prostate tumor growth and metastasis and is associated with increased intratumor infiltration of macrophages. We further demonstrate that prostate epithelial RON loss induces transcriptional reprogramming of macrophages to support expression of classical M1 markers and suppress expression of alternative M2 markers. Interestingly, our results show epithelial RON activation drives upregulation of RON expression in macrophages as a positive feed-forward mechanism to support prostate tumor growth. Using 3D coculture assays, we provide additional evidence that epithelial RON expression coordinates interactions between prostate tumor cells and macrophages to promote macrophage-mediated tumor cell growth. Taken together, our results suggest that RON receptor signaling in prostate tumor cells directs the functions of macrophages in the prostate tumor microenvironment to promote prostate cancer. IMPLICATIONS: Epithelial RON is a novel immunotherapeutic target that is responsible for directing the macrophage antitumor immune response to support prostate tumor growth and progression.

Mammary-specific Ron receptor overexpression induces highly metastatic mammary tumors associated with beta-catenin activation.

Activated growth factor receptor tyrosine kinases (RTK) play pivotal roles in a variety of human cancers, including breast cancer. Ron, a member of the Met RTK proto-oncogene family, is overexpressed or constitutively active in 50% of human breast cancers. To define the significance of Ron overexpression and activation in vivo, we generated transgenic mice that overexpress a wild-type or constitutively active Ron receptor in the mammary epithelium. In these animals, Ron expression is significantly elevated in mammary glands and leads to a hyperplastic phenotype by 12 weeks of age. Ron overexpression is sufficient to induce mammary transformation in all transgenic animals and is associated with a high degree of metastasis, with metastatic foci detected in liver and lungs of >86% of all transgenic animals. Furthermore, we show that Ron overexpression leads to receptor phosphorylation and is associated with elevated levels of tyrosine phosphorylated beta-catenin and the up-regulation of genes, including cyclin D1 and c-myc, which are associated with poor prognosis in patients with human breast cancers. These studies suggest that Ron overexpression may be a causative factor in breast tumorigenesis and provides a model to dissect the mechanism by which the Ron induces transformation and metastasis.

The Ron receptor tyrosine kinase regulates acute lung injury and suppresses nuclear factor kappaB activation.

Emerging information implies that the Ron receptor tyrosine kinase may play a role in the inflammatory response. However, the manner in which this receptor contributes to the response is not well understood. In the present studies, we investigated the role of the Ron receptor in the acute lung inflammatory response. Wild-type and mutant mice lacking the tyrosine kinase domain of Ron (Ron TK-/-) were subjected to acute lung injury induced by intranasal administration of bacterial lipopolysaccharide (LPS). Wild-type mice showed increased lung injury after LPS administration, as determined by the leakage of albumin into the lung and by histopathological changes. Ron TK-/- mice had more than twice the amount of albumin leak and much greater thickening of the alveolar septae. Lipopolysaccharide administration caused neutrophil recruitment into the lungs, as measured by myeloperoxidase. However, Ron TK-/- mice had much higher baseline levels of myeloperoxidase, which did not increase further after LPS. Lung injury in wild-type mice occurred with activation of the transcription factor, nuclear factor kappaB (NF-kappaB), and subsequent increases in intrapulmonary generation of tumor necrosis factor alpha. In TK-/- mice, there was far less IkappaB-alpha and IkappaB-beta protein and greater activation of NF-kappaB. This was associated with substantially increased production of tumor necrosis factor alpha and the nitric oxide (NO) by-product, nitrite. The data suggest that the Ron receptor tyrosine kinase plays an important regulatory role in acute inflammatory lung injury by suppressing signals leading to activation of NF-kappaB.

HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses.

The Ron receptor is overexpressed in human breast cancers and is associated with heightened metastasis and poor survival. Ron overexpression in the mammary epithelium of mice is sufficient to induce aggressive mammary tumors with a high degree of metastasis. Despite the well-documented role of Ron in breast cancer, few studies have examined the necessity of the endogenous Ron ligand, hepatocyte growth factor-like protein (HGFL) in mammary tumorigenesis. Herein, mammary tumor growth and metastasis were examined in mice overexpressing Ron in the mammary epithelium with or without HGFL. HGFL ablation decreased oncogenic Ron activation and delayed mammary tumor initiation. HGFL was important for tumor cell proliferation and survival. HGFL loss resulted in increased numbers of macrophages and T-cells within the tumor. T-cell proliferation and cytotoxicity dramatically increased in HGFL deficient mice. Biochemical analysis of HGFL proficient tumors showed increased local HGFL production, with HGFL loss decreasing ß-catenin expression and NF-κB activation. Re-expression of HGFL in HGFL deficient tumor cells stimulated cell migration and invasion with coordinate activation of NF-κB and reduced apoptosis. Together, these results demonstrate critical in vivo functions for HGFL in promoting breast tumorigenesis and suggest that targeting HGFL may inhibit tumor growth and reactivate anti-tumor immune responses.

Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic LXXLL peptide antagonists that target the activation domains of the vitamin D and retinoid X receptors.

The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through its ability to regulate cellular programs of gene expression. Although RXR appears to participate as a heterodimeric partner with the VDR, absolute evidence for its role remains equivocal in vivo. To test this role and to investigate the requirement for comodulator interaction, we identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. We used a mammalian cell two-hybrid system to screen a series of nuclear receptor (NR)-reactive LXXLL peptides previously identified through phage display screening for hormone-dependent reactivity with either VDR or RXR. Three categories of peptides were identified: those reactive with both VDR and RXR, those selective for RXR, and those unreactive to either receptor. Peptide fusion proteins were then examined in MC3T3-E1 cells for their ability to block induction of the osteocalcin (OC) promoter by 1,25(OH)2D3 or stimulation of a retinoic acid response element-thymidine kinase (RARE-TK) reporter by 9-cis-RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)2D3-dependent transcription by up to 75%. Control LXXLL sequences derived from Src-1 and Grip also suppressed 1,25(OH)2D3-induced transactivation; peptides that interacted with RXR blocked 9-cis-RA-induced transcription. Interestingly, two RXR-interacting peptides were also found to block 1,25(OH)2D3 response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)2D3-induced OC gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)2D3.

The role of calcium in hypoxia-induced signal transduction and gene expression.

Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors.

Genomic and physiological analysis of oxygen sensitivity and hypoxia tolerance in PC12 cells.

The mechanisms by which cells adapt and respond to changes in oxygen tension remain largely unknown. Our laboratory has used the PC12 cell line to study both biophysical and molecular responses to hypoxia. This chapter summarizes our findings. We found that membrane depolarization that occurred when PC12 cells were exposed to reduced O(2) was mediated by a specific potassium channel, the Kv1.2 channel. The membrane depolarization leads to increased Ca(2+) conductance through a voltage-sensitive channel, which in turn mediates the release of the neurotransmitters dopamine, adenosine, glutamate, and GABA. In addition, increased intracellular Ca(2+) and other signaling systems regulate hypoxia-induced gene expression, which contributes to the adaptive response to reduced O(2+). We identified several critical signaling pathways that regulate a complex gene expression profile in PC12 cells during hypoxia. These include the cAMP-protein kinase A, Ca(2+)-calmodulin, p42/44 mitogen-activated protein kinase (MAPK), stress-activated protein kinase (SAPK; p38 kinase), and the phosphatidylinositol 3-kinase-AKT as regulators of gene expression. Several of these pathways regulate hypoxia-specific transcription factors that are members of the hypoxia-inducible factor (HIF) family. Recently, we have successfully used subtractive cDNA libraries and microarray analysis to identify the genomic profile that mediates the cellular response to hypoxia.

Hepatocyte growth factor-like protein is required for prostate tumor growth in the TRAMP mouse model.

The Ron receptor is deregulated in a variety of cancers. Hepatocyte growth factor-like protein (HGFL) is the ligand for Ron and is constitutively secreted from hepatocytes into the circulation. While a few recent reports have emerged analyzing ectopic HGFL overexpression of in cancer cells, no studies have examined host-produced HGFL in tumorigenesis. To examine HGFL function in prostate cancer, the TRAMP mouse model, which is predisposed to develop prostate tumors, was utilized. Prostate tumors from TRAMP mice exhibit elevated levels of HGFL, which correlated with upregulation in human prostate cancer. To directly implicate HGFL in prostate tumorigenesis, TRAMP mice deficient in HGFL (HGFL-/-TRAMP+) were generated. HGFL-/- TRAMP+ mice developed significantly smaller prostate tumors compared to controls. Analysis of HGFL-/- tumors revealed reduced tumor vascularization. No differences in cancer cell proliferation were detected between HGFL-/- TRAMP+ and HGFL+/+ TRAMP+ mice. However, a significant increase in cancer cell death was detected in HGFL-/- TRAMP+ prostates which correlated with decreased pro-survival targets. In vitro analysis demonstrated robust STAT3 activation resulting in Bcl2-dependent survival following treatment of prostate cancer cells with HGFL. These data document a novel function for endogenous HGFL in prostate cancer by imparting a critical survival signal to tumor cells.

The use of pharmacogenomics for selection of therapy in non-small-cell lung cancer.

INTRODUCTION: Performance status (PS) is the only known clinical predictor of outcome in patients with advanced non-small-cell lung cancer (NSCLC), although pharmacogenomic markers may also correlate with outcome. The aim of our study was to correlate clinical and pharmacogenomic measures with overall survival. METHODS: This was an IRB approved, retrospective study in which the medical records of 50 patients with advanced NSCLC from 1998-2008 were reviewed, and gender, race, PS, and chemotherapy regimens were documented. Stromal expression of pharmacogenomic markers (VEGFR, ERCC1, 14-3-3σ, pAKT, and PTEN) was measured. Clinical factors and pharmacogenomics markers were compared to overall survival using a Cox proportional hazards model. RESULTS: Forty patients received platinum-based therapy. Median age was 65 years. Improved PS, female gender, and gemcitabine therapy were significantly associated with longer overall survival (P = 0.004, P = 0.04, and P = 0.003, respectively). Age was not associated with survival. Caucasians had better overall survival in comparison to African Americans with median survival of 14.8 months versus 10.4 months (P = 0.1). Patients treated with platinum-based therapy had better survival of 15 months versus 8 months for non-platinum based therapy (P = 0.01). There was no significant association between any of the pharmacogenomics markers and overall survival other than in patients treated with platinum, in whom ERCC1 negativity was strongly associated with longer survival (P = 0.007). CONCLUSION: ERCC1 negativity with platinum therapy, gemcitabine therapy, good PS, and female gender all correlated with improved overall survival in patients with advanced NSCLC.

Myeloid-specific expression of Ron receptor kinase promotes prostate tumor growth.

Ron receptor kinase (MST1R) is important in promoting epithelial tumorigenesis, but the potential contributions of its specific expression in stromal cells have not been examined. Herein, we show that the Ron receptor is expressed in mouse and human stromal cells of the prostate tumor microenvironment. To test the significance of stromal Ron expression, prostate cancer cells were orthotopically implanted into the prostates of either wild-type or Ron tyrosine kinase deficient (TK(-/-); Mst1r(-/-)) hosts. In TK(-/-) hosts, prostate cancer cell growth was significantly reduced as compared with tumor growth in TK(+/+) hosts. Prostate tumors in TK(-/-) hosts exhibited an increase in tumor cell apoptosis, macrophage infiltration and altered cytokine expression. Reciprocal bone marrow transplantation studies and myeloid cell-specific ablation of Ron showed that loss of Ron in myeloid cells is sufficient to inhibit prostate cancer cell growth. Interestingly, depletion of CD8(+) T cells, but not CD4(+) T cells, was able to restore prostate tumor growth in hosts devoid of myeloid-specific Ron expression. These studies show a critical role for the Ron receptor in the tumor microenvironment, whereby Ron loss in tumor-associated macrophages inhibits prostate cancer cell growth, at least in part, by derepressing the activity of CD8(+) T cells.

Clinical and molecular predictors of recurrence in stage I non-small cell lung cancer.

BACKGROUND: Patients with stage I lung cancer undergoing a complete resection have a 25% risk of recurrence. Factors predictive for recurrence are critically needed. In the present study, we prospectively examined clinical and molecular factors that may predict a poor outcome. METHODS: Patients with stage I non-small cell lung cancer undergoing surgical resection were enrolled into an institutional registry. Clinical demographics and outcomes data were prospectively collected. Patients who received neoadjuvant therapy or patients who died within 30 days of surgery were excluded from this analysis. Molecular factors involved in cell proliferation, cell cycle control, apoptosis, and angiogenesis were analyzed. The primary endpoint was recurrence-free survival. RESULTS: One hundred and two patients were enrolled between March 2006 and April 2009. There were 25 (25%) documented recurrences. In univariate analysis, male sex, increased tumor standard uptake value, tumor size, final pathology stage, arterial invasion, percent nuclear phosphorylated AKT, vascular endothelial growth factor score, negative cyclin D1 protein expression, and percent nuclear cyclin D1 expression were predictive of decreased recurrence-free survival. All factors with a p value of 0.1 or less were included in multivariate analysis. Male sex, final pathology stage, vascular endothelial growth factor score, and percent nuclear cyclin D1 expression were significant independent predictors for poor prognosis. CONCLUSIONS: Four clinical and molecular factors were associated with prognosis in a prospective study of stage I non-small cell lung cancer.

Protein kinase Czeta represses the interleukin-6 promoter and impairs tumorigenesis in vivo.

Gene alterations in tumor cells that confer the ability to grow under nutrient- and mitogen-deficient conditions constitute a competitive advantage that leads to more-aggressive forms of cancer. The atypical protein kinase C (PKC) isoform, PKCzeta, has been shown to interact with the signaling adapter p62, which is important for Ras-induced lung carcinogenesis. Here we show that PKCzeta-deficient mice display increased Ras-induced lung carcinogenesis, suggesting a new role for this kinase as a tumor suppressor in vivo. We also show that Ras-transformed PKCzeta-deficient lungs and embryo fibroblasts produced more interleukin-6 (IL-6), which we demonstrate here plays an essential role in the ability of Ras-transformed cells to grow under nutrient-deprived conditions in vitro and in a mouse xenograft system in vivo. We also show that PKCzeta represses histone acetylation at the C/EBPbeta element in the IL-6 promoter. Therefore, PKCzeta, by controlling the production of IL-6, is a critical signaling molecule in tumorigenesis.

Homozygous K5Cre transgenic mice have wavy hair and accelerated malignant progression in a murine model of skin carcinogenesis.

Mice with conditional gene deletions have been extremely valuable in allowing investigators to study the genes of interest in a tissue-specific manner. The Cre-loxP recombination system provides a powerful tool to produce targeted rearrangements of particular genes. The keratin 5-Cre recombinase (K5Cre) transgenic mouse line has been used to generate skin specific gene deletions. We found that the K5Cre mice display a unique phenotype when bred to homozygosity. The K5Cre(+/+) mice have a wavy hair coat and curly whiskers. Histologically, the hair follicles appear disoriented. Over time, the K5Cre(+/+) mice develop patches of alopecia. These mice are also runted when compared to wild-type controls. Fostering the K5Cre(+/+) pups to wild-type mothers results in normal weight gain, suggesting a maternal defect in milk production. When the K5Cre(+/+) mammary glands were examined, we not only found a significant decrease in the number of mammary branches in the virgin females, but also a greater number of quiescent alveoli units in the lactating glands. When the K5Cre(+/+) mice were bred to v-Ha-ras (Tg . AC) transgenic mice, the resulting Tg . AC(+/-) K5Cre(+/+) offspring were utilized in a chemically induced skin carcinogenesis model. The mice were treated with 2.5 microg of 12-O-tetradecanoylphorbol-13-acetate (TPA) weekly for 10 wk. No difference was observed in the time to onset of papilloma formation, the number of papillomas and the average papilloma volume between the Tg . AC(+/-) K5Cre(+/+) mice and their corresponding controls. Surprisingly, however, the K5Cre(+/+) papillomas displayed an accelerated tendency to malignant progression; in addition, the frequency of malignant transformation of the papillomas is significantly enhanced. Although the K5Cre(+/+) mice resemble waved-1 and -2 mutants, the molecular basis for the K5Cre(+/+) phenotype is probably different. In conclusion, we discovered a unique phenotype associated with the K5Cre(+/+) transgenic line.