Development of a rational strategy for integration of lactate dehydrogenase A suppression into therapeutic algorithms for head and neck cancer.

BACKGROUND: Lactate dehydrogenase (LDH) is a critical metabolic enzyme. LDH A (LDHA) overexpression is a hallmark of aggressive malignancies and has been linked to tumour initiation, reprogramming and progression in multiple tumour types. However, successful LDHA inhibition strategies have not materialised in the translational and clinical space. We sought to develop a rational strategy for LDHA suppression in the context of solid tumour treatment. METHODS: We utilised a doxycycline-inducible short hairpin RNA (shRNA) system to generate LDHA suppression. Lactate and LDH activity levels were measured biochemically and kinetically using hyperpolarised 13C-pyruvate nuclear magnetic resonance spectroscopy. We evaluated effects of LDHA suppression on cellular proliferation and clonogenic survival, as well as on tumour growth, in orthotopic models of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), alone or in combination with radiation. RESULTS: shRNA suppression of LDHA generated a time-dependent decrease in LDH activity with transient shifts in intracellular lactate levels, a decrease in carbon flux from pyruvate into lactate and compensatory shifts in metabolic flux in glycolysis and the Krebs cycle. LDHA suppression decreased cellular proliferation and temporarily stunted tumour growth in ATC and HNSCC xenografts but did not by itself result in tumour cure, owing to the maintenance of residual viable cells. Only when chronic LDHA suppression was combined with radiation was a functional cure achieved. CONCLUSIONS: Successful targeting of LDHA requires exquisite dose and temporal control without significant concomitant off-target toxicity. Combinatorial strategies with conventional radiation are feasible as long as the suppression is targeted, prolonged and non-toxic.

Dysregulated Krüppel-like factor 4 and vitamin D receptor signaling contribute to progression of hepatocellular carcinoma.

BACKGROUND & AIMS: Krüppel-like factor 4 (KLF4) is a transcription factor and putative tumor suppressor. However, little is known about its effects in hepatocellular carcinogenesis. We investigated the clinical significance, biologic effects, and mechanisms of dysregulated KLF4 signaling. METHODS: We performed microarray analysis of hepatocellular carcinoma (HCC) tissues. We used molecular biology analyses and animal models to evaluate activation and function of KLF4-vitamin D receptor (VDR) pathway. RESULTS: Expression of KLF4 protein was decreased or lost in primary HCC samples, in particular, lymph node metastases, compared with normal liver tissues. Loss of KLF4 from primary tumors was significantly associated with reduced survival time and was identified as a prognostic marker. Most human HCC cell lines had losses or substantial decreases in levels of KLF4. Exogenous expression of KLF4 in HCC cells upregulated expression of mesenchymal-epithelial transition (MET) and inhibited their migration, invasion, and proliferation in vitro. When these cells were injected into mice, tumors grew more slowly and metastasis was inhibited, compared with HCC cells that did not express KLF4. VDR is a direct transcriptional target of KLF4; we identified 2 sites in the VDR promoter that bound specifically to KLF4. Increased expression of VDR sensitized tumor cells to the inhibitory effects of vitamin D. CONCLUSIONS: KLF4 binds to the promoter of VDR to regulate its expression; levels of KLF4 are reduced and levels of VDR are increased in HCC cell lines and primary tumor samples. Expression of KLF4 in HCC cells sensitizes them to the anti-proliferative effects of VD3. This pathway might be manipulated to prevent or treat liver cancer.

Disruption of Klf4 in villin-positive gastric progenitor cells promotes formation and progression of tumors of the antrum in mice.

BACKGROUND & AIMS: Krüppel-like factor 4 (Klf4) is a putative gastric tumor suppressor gene. Rare, villin-positive progenitor cells in the gastric antrum have multilineage potential. We investigated the function of Klf4 in these cells and in gastric carcinogenesis. METHODS: We created mice with disruption of Klf4 in villin-positive antral mucosa cells (Villin-Cre(+);Klf4(fl/fl) mice). Villin-Cre(+);Klf4(fl/fl) and control mice were given drinking water with or without 240 ppm N-methyl-N-nitrosourea at 5 weeks of age and thereafter on alternating weeks for a total of 10 weeks. Gastric mucosa samples were collected at 35, 50, or 80 weeks of age from mice that were and were not given N-methyl-N-nitrosourea, and analyzed by histopathologic and molecular analyses. Findings were compared with those from human gastric tumor specimens. RESULTS: Preneoplasia formed progressively in the antrum in 35- to 80-week-old Villin-Cre(+);Klf4(fl/fl) mice. Gastric tumors developed in 29% of 80-week-old Villin-Cre(+);Klf4(fl/fl) mice, which were located exclusively in the lesser curvature of the antrum. N-methyl-N-nitrosourea accelerated tumor formation, and tumors developed significantly more frequently in Villin-Cre(+);Klf4(fl/fl) mice than in control mice, at 35 and 50 weeks of age. Mouse and human gastric tumors had reduced expression of Krüppel-like factor 4 and increased expression of FoxM1 compared with healthy gastric tissue. Expression of Krüppel-like factor 4 suppressed transcription of FoxM1. CONCLUSIONS: Inactivation of Klf4 in villin-positive gastric progenitor cells induces transformation of the gastric mucosa and tumorigenesis in the antrum in mice. Villin-Cre(+);Klf4(fl/fl) have greater susceptibility to chemical-induced gastric carcinogenesis and increased rates of gastric tumor progression than control mice.

KLF4α up-regulation promotes cell cycle progression and reduces survival time of patients with pancreatic cancer.

BACKGROUND & AIMS: Krüppel-like factor 4 (KLF4) is a transcription factor associated with tumor suppression and oncogenesis. KLF4 suppresses pancreatic tumorigenesis by unknown mechanisms; we investigated alterations that might affect KLF4 function and lead to tumor formation. METHODS: We identified different isoforms of KLF4 in pancreatic cancer cells by reverse-transcriptase polymerase chain reaction, cloning, and DNA sequence analyses. We constructed vectors to express the isoform KLF4α and characterize its function. Using real-time polymerase chain reaction, immunoprecipitation, and immunohistochemical analyses, we assessed expression of KLF4α in pancreatic cancer cell lines and tumor tissue samples; xenograft models were used to determine the effect of KLF4α on pancreatic tumorigenesis. RESULTS: We identified 4 KLF4 isoforms in human pancreatic cancer cells, designated KLF4α, KLF4ß, KLF4γ, and KLF4δ. KLF4α localized primarily to the cytoplasm; its protein and messenger RNA were up-regulated in pancreatic cancer cell lines with high metastatic potential and human pancreatic tumors compared with normal pancreatic tissue. Transgenic expression of KLF4α reduced expression of p27(Kip1) and p21(Cip1), promoting cell cycle progression and in vivo tumor formation by pancreatic cancer cells. Increased expression of KLF4α in pancreatic tumor tissue was inversely correlated with overall time of survival in patients with stage II pancreatic ductal adenocarcinoma. CONCLUSIONS: We identified a splice variant of KLF4 (KLF4α) that is up-regulated in aggressive pancreatic cancer cells and human pancreatic tumor tissues. Increased expression promotes growth of pancreatic tumors in mice and is associated with reduced survival times of patients.

Molecular basis of the synergistic antiangiogenic activity of bevacizumab and mithramycin A.

The impact of antiangiogenic therapy on the Sp1/vascular endothelial growth factor (VEGF) pathway and that of alteration of Sp1 signaling on the efficacy of antiangiogenic therapy is unclear, yet understanding their interactions has significant clinical implications. Treatment with bevacizumab, a neutralizing antibody against VEGF, suppressed human pancreatic cancer growth in nude mice. Gene expression analyses revealed that this treatment substantially up-regulated the expression of Sp1 and its downstream target genes, including VEGF and epidermal growth factor receptor, in tumor tissues, whereas it did not have this effect on pancreatic cancer cells in culture. Treatment with mithramycin A, an Sp1 inhibitor, suppressed the expression of Sp1 and its downstream target genes in both cell culture and tumors growing in nude mice. Combined treatment with bevacizumab and mithramycin A produced synergistic tumor suppression, which was consistent with suppression of the expression of Sp1 and its downstream target genes. Thus, treatment with bevacizumab may block VEGF function but activate the pathway of its expression via positive feedback. Given the fact that Sp1 is an important regulator of the expression of multiple angiogenic factors, bevacizumab-initiated up-regulation of Sp1 and subsequent overexpression of its downstream target genes may profoundly affect the potential angiogenic phenotype and effectiveness of antiangiogenic strategies for human pancreatic cancer. Therefore, this study is the first to show the significance and clinical implications of alteration of Sp1 signaling in antiangiogenic therapy for pancreatic cancer and other cancers.

Inhibition of the growth and metastasis of human colon cancer by restoration of RUNX3 expression in cancer cells.

Recent studies demonstrated an epigenetic inactivation of the runt-related transcription factor 3 (RUNX3) gene in human colon cancer. However, it remains unclear whether RUNX3 is tumor suppressive in colon cancer and, if so, the underlying molecular mechanisms of this activity are still unknown. In the present study, we sought to determine the level of RUNX3 expression in human colon tumor specimens and used an animal model of colon cancer to determine the impact of RUNX3 expression on tumor growth and metastasis. First, we analyzed RUNX3 expression in 83 human colon tumor specimens using immunohistochemical, reverse transcriptase-polymerase chain reaction, and Western blot analysis. RUNX3 mRNA and protein expression levels were consistently lower in tumor tissue specimens than in matched normal colon tissue specimens. Also, restoration of RUNX3 expression in colon cancer cells using gene transfer inhibited colon tumor growth and metastasis in our animal model, which was consistent with inhibition of colon tumor growth in vitro. Collectively, our clinical and experimental data support the notion that RUNX3 is a tumor suppressor in human colon cancer.

RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer.

PURPOSE: Recent studies indicated that RUNX3 exhibits potent antitumor activity. However, the underlying molecular mechanisms of this activity remain unclear. In the present study, we used a gastric cancer model to determine the effect of RUNX3 expression on tumor angiogenesis. EXPERIMENTAL DESIGN: The effects of increased RUNX3 expression on vascular endothelial growth factor (VEGF) expression in and angiogenic potential of human gastric cancer cells were determined in vitro and in animal models. RUNX3 and VEGF expression was determined in 120 human gastric cancer specimens and their relationship was analyzed. RESULTS: RUNX3 gene transfer suppressed VEGF expression in human gastric cancer cells. Down-regulation of VEGF expression correlated with a significantly impaired angiogenic potential of human gastric cancer cells. Furthermore, RUNX3 restoration inhibited tumor growth and metastasis in animal models, which was consistent with inhibition of angiogenesis as determined by evaluating VEGF expression and tumor microvessel formation. In gastric cancer specimens, loss or decrease in RUNX3 expression inversely associated with increased VEGF expression and elevated microvessel formation. CONCLUSIONS: Our clinical and experimental data provide a novel molecular mechanism for the antitumor activity of RUNX3 and may help design effective therapy targeting RUNX3 pathway to control gastric cancer growth and metastasis.

Loss of Krüppel-like factor 4 expression contributes to Sp1 overexpression and human gastric cancer development and progression.

PURPOSE: Increasing evidence indicates that the transcription factor, Sp1, regulates the expression of multiple genes involved in tumor development and progression. We have recently reported that Sp1 overexpression is directly correlated with the angiogenic potential of and poor prognosis for human gastric cancer. However, the underlying mechanisms that result in Sp1 overexpression remain unclear. EXPERIMENTAL DESIGN: The expression of Sp1 and Krüppel-like factor 4 (KLF4), a potential tumor suppressor gene, in gastric cancer tissue was analyzed by immunohistochemistry and Western blot analysis. Alterations of Sp1 and KLF4 expression were achieved by gene transfer and verified by Northern and Western blot analyses. Furthermore, Sp1 promoter activity assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay were done to identify the KLF4 binding sites on the Sp1 promoter. RESULTS: Mutually exclusive expression of Sp1 and KLF4 was evident in gastric cancer and noncancerous tissue. Specifically, strong Sp1 expression but loss of KLF4 expression was found in cancer tissue, whereas the adjacent noncancerous tissue showed negative Sp1 expression but strong KLF4 expression. Enforced KLF4 expression repressed Sp1 expression at the promoter activity, mRNA, and protein levels. Moreover, a region within the proximal Sp1 promoter was identified to have overlapping KLF4- and Sp1-binding sites, to which KLF4 and Sp1 compete for binding. Sp1 positively regulated its own promoter, whereas KLF4 did the opposite. CONCLUSIONS: Our data suggests that disruption of KLF4-mediated negative regulation contributes to the molecular events of Sp1 overexpression and to the development and progression of human gastric cancer.

Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.

Identification of precise prognostic marker and effective therapeutic target is pivotal in the treatment of gastric cancer. In the present study, we determined the level of RUNX3 expression in gastric cancer cells and gastric cancer specimens and the impact of its alteration on cancer biology and clinical outcome. There was a loss or substantial decrease of RUNX3 protein expression in 86 cases of gastric tumors as compared with that in normal gastric mucosa (P < 0.0001), which was significantly associated with inferior survival duration (P = 0.0005). In a Cox proportional hazards model, RUNX3 expression independently predicted better survival (P = 0.036). Moreover, various human gastric cancer cell lines also exhibited loss or drastic decrease of RUNX3 expression. Enforced restoration of RUNX3 expression led to down-regulation of cyclin D1 but to up-regulation of p27, caspase 3, 7, and 8 expression, cell cycle arrest, and apoptosis in vitro, and dramatic attenuation of tumor growth and abrogation of metastasis in animal models. Therefore, we offered both clinical and mechanistic evidence that RUNX3 was an independent prognostic factor and a potential therapeutic target for gastric cancer.

Direct demonstration of negative regulation of tumor growth and metastasis by host-inducible nitric oxide synthase.

Inducible nitric oxide synthase (NOS) II expression can be induced in the tumor bed, predominantly in host cells that infiltrate and surround a tumor. However, the impact of this physiological NOS II expression in host cells on tumor growth and metastasis remains unclear because of a lack of appropriate experimental approaches. In the present study, three NOS II-null (NOS II(-/-)) tumor cell lines, KX-dw1, KX-dw4, and KX-dw7, were established and verified using Southern, Northern, and Western blot analysis, and nitric oxide production assays. Cells from these lines were then s.c. and i.v. injected into NOS II(+/+) and NOS II(-/-) C57BL/6 mice. NOS II protein expression and enzyme activity were clearly detected in the tumors that formed in NOS II(+/+) mice but not in those that formed in NOS II(-/-) mice. Consistent with the absence of NOS II expression in the tumor stroma, KX-dw1, KX-dw4, and KX-dw7 cells grew much faster and produced many more experimental lung metastases in NOS II(-/-) mice than in NOS II(+/+) mice. Therefore, physiological expression of NOS II in host cells directly inhibits tumor growth and metastasis.

Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis.

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein-DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.

Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer.

The transcription factor Sp1 regulates the expression of multiple genes. However, its expression and role in human tumor development and progression remain unclear. Using immunohistochemistry, we investigated Sp1 expression patterns in 86 cases of human gastric cancer having various clinicopathologic characteristics, 57 normal gastric tissue specimens, and 53 lymph node metastases. We found that Sp1 protein was expressed predominantly in the nuclei of cells located in the mucous neck region, whereas Sp1 expression was not detected either in the cells located toward the gastric pit (foveolar differentiation) or cells of the glandular epithelium (glandular differentiation). In sharp contrast, strong Sp1 expression was detected in tumor cells, whereas no or very weak Sp1 expression was detected in stromal cells and normal glandular cells surrounding or within the tumors. We also evaluated the effect of Sp1 expression on the survival of patients who have undergone surgical resection. The median survival duration in patients who had a tumor with negative, weak, and strong Sp1 expression was 43, 37, and 8 months, respectively (P = 0.0075). Next, Sp1 expression, stage, completeness of resection, age, and sex were entered into a Cox proportional hazard model. In multivariate analysis, Sp1 (P = 0.003) and stage (P < 0.001) were independently prognostic of survival. Therefore, normal and malignant gastric tissues have unique Sp1 expression patterns. Given the importance of Sp1 in the expression of multiple molecules key to tumor cell survival, growth, and angiogenesis, its disregulated expression and activation may play important roles in gastric cancer development and progression.

Regulation of receptor tyrosine kinases by miRNA: overexpression of miRNA using lentiviral inducible expression vectors.

MicroRNAs have the ability to alter and regulate multiple genes, including RTK family members, making them an attractive approach for molecular therapeutic development. We use a pCDNA6.2-EmGFP-microRNA expression vector to overexpress individual mature microRNA and then transfer the expression cassette into a single, inducible lentiviral vector (pINDUCER20). We successfully use this system to create a pINDUCER-EmGFP-miRNA27a expression vector and generate a stable head and neck cancer cell line (UM-SCC-22A) that inducibly expresses miRNA-27a, resulting in targeted epidermal growth factor receptor down regulation. In this chapter, we describe the protocol for engineering the pINDUCER-EmGFP-microRNA expression vector, producing lentiviral particles for target cell infection, and evaluating downregulation of gene expression.

A high expression level of insulin-like growth factor I receptor is associated with increased expression of transcription factor Sp1 and regional lymph node metastasis of human gastric cancer.

Insulin-like growth factor I receptor (IGF-IR) is critical to cell survival and growth and altered IGF-IR expression is found in many human cancers. However, its expression and potential role in gastric cancer development and progression has not been explored. The IGF-IR expression level was determined via immunohistochemistry in primary tumor and lymph node metastasis of 86 cases of resected gastric cancer. Relationships of IGF-IR expression with transcription factor Spl expression and clinicopathological features were analyzed. The impact of altered Sp1 expression on IGF-IR expression and gastric cancer biology was further determined using small inhibitory RNA for Sp1 mRNA. We found that IGF-IR was overexpressed in 62% of the tumor samples when compared with adjacent tumor-free gastric mucosa. Patients with lymph node metastases had strong expression of IGF-IR in both primary and metastatic tumor cells. IGF-IR overexpression in the primary tumor correlated with increased lymph node metastasis. Furthermore, the level of IGF-IR expression directly correlated with that of Spl, an important transcription factor for IGF-IR regulation. Knocking-down of Spl expression by small inhibitory RNA led to decreased IGF-IR expression and attenuated growth and metastasis of gastric cancer cells. Therefore, dysregulated expression of IGF-IR and/or Sp1 may contribute to the growth and metastasis of gastric cancer and potentially can be a target of therapeutic intervention.

Mouse Models of Metastatic Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma is a deadly disease. Its etiology is unknown, and metastatic disease kills themajority of patients who have it. Effective prevention is clearly the ultimate goal for eradicating this diseaseprovided that the effects of environmental and genetic elements on pancreatic cancer development arefully understood. Currently, it appears that the control of pancreatic cancer metastasis is of immediateurgency. Fulfillment of this difficult task relies on knowledge of the cellular and molecular biology of metastasis.The use of relevant animal models will help define each aspect of this complicated process.

A Novel, Clinically Relevant Animal Model of Metastatic Pancreatic Adenocarcinoma Biology and Therapy.

In this study, we report a metastatic model of Panc02 murine pancreatic adenocarcinoma. Parental Panc02cells were orthotopically implanted into the pancreas of syngeneic C57BL/6 mice. Tumor cells were isolatedfrom liver micrometastases 90 d after tumor implantation and established as a culture (Panc02-H1).The Panc02-H1 cells were then implanted into the pancreas of mice. Liver metastases were then collectedand established as Panc02-H2 cells. This process was repeated until the Panc02-H7 cell line was established.These cells were extremely aggressive after implantation as manifested by progressive growth in the pancreas,peritoneal dissemination, and distant metastasis to multiple organs, including the liver and lungs.Moreover, Panc02-H7 cells expressed the inducible nitric oxide synthase gene at a very low level in cultureand produced highly vascularized tumors having a large number of infiltrating macrophages. Collectively,this model system should be a valuable tool for investigating the molecular mechanisms governing pancreaticcancer growth and metastasis and exploring potential treatment modalities for this disease.

The critical role of dysregulated FOXM1-PLAUR signaling in human colon cancer progression and metastasis.

PURPOSE: The mammalian Forkhead Box (Fox) transcription factor FOXM1 is implicated in tumorigenesis including mouse intestinal cancer. However, the clinical significance of FOXM1 signaling in human colorectal cancer pathogenesis remains unknown. EXPERIMENTAL DESIGN: We investigated FOXM1 expression in 203 cases of primary colon cancer and matched normal colon tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on colon cancer growth and metastasis using in vitro and animal models of colon cancer. RESULTS: We found weak expression of FOXM1 protein in the colon mucosa, whereas we observed strong FOXM1 expression in tumor-cell nuclei of colon cancer and lymph node metastases. A Cox proportional hazards model revealed that FOXM1 expression was an independent prognostic factor in multivariate analysis. Experimentally, overexpression of FOXM1 by gene transfer significantly promoted the growth and metastasis of colon cancer cells in orthotopic mouse models, whereas knockdown of FOXM1 expression by siRNA did the opposite. Promotion of colon tumorigenesis by FOXM1 directly and significantly correlated with activation of urokinase-type plasminogen activator receptor (PLAUR) expression and elevation of invasion and metastasis. CONCLUSIONS: Given the importance of FOXM1 in regulation of the expression of genes key to cancer biology, dysregulated expression and activation of FOXM1 may play important roles in colon cancer progression and metastasis.

Dysregulated expression of FOXM1 isoforms drives progression of pancreatic cancer.

The transcription factor Forkhead box M1 (FOXM1) plays important roles in oncogenesis. However, the expression statuses of FOXM1 isoforms and their impact on and molecular basis in oncogenesis are unknown. We sought to determine the identities of FOXM1 isoforms in and the impact of their expression on pancreatic cancer development and progression using human tissues, cell lines, and animal models. Overexpression of FOXM1 mRNA and protein was pronounced in human pancreatic tumors and cancer cell lines. We identified five FOXM1 isoforms present in pancreatic cancer: FOXM1a, FOXM1b, and FOXM1c along with two isoforms tentatively designated as FOXM1b1 and FOXM1b2 because they were closely related to FOXM1b. Interestingly, FOXM1c was predominantly expressed in pancreatic tumors and cancer cell lines, whereas FOXM1a expression was generally undetectable in them. Functional analysis revealed that FOXM1b, FOXM1b1, FOXM1b2, and FOXM1c, but not FOXM1a, promoted pancreatic tumor growth and metastasis. Consistently, FOXM1b, FOXM1b1, FOXM1b2, and FOXM1c activated transcription of their typical downstream genes. Also, Sp1 mechanistically activated the FOXM1 promoter, whereas Krüppel-like factor 4 (KLF4) repressed its activity. Finally, we identified an Sp1- and KLF4-binding site in the FOXM1 promoter and showed that both Sp1 and KLF4 protein bound directly to it. Deletion mutation of this binding site significantly attenuated the transcriptional regulation of the FOXM1 promoter positively by Sp1 and negatively by KLF4. We showed that overexpression of specific FOXM1 isoforms critically regulates pancreatic cancer development and progression by enhancing tumor cell invasion and metastasis. Our findings strongly suggest that targeting specific FOXM1 isoforms effectively attenuates pancreatic cancer development and progression.

A novel FoxM1-caveolin signaling pathway promotes pancreatic cancer invasion and metastasis.

Caveolin-1 (Cav-1), a principal structural component of caveolar membrane domains, contributes to cancer development but its precise functional roles and regulation remain unclear. In this study, we determined the oncogenic function of Cav-1 in preclinical models of pancreatic cancer and in human tissue specimens. Cav-1 expression levels correlated with metastatic potential and epithelial-mesenchymal transition (EMT) in both mouse and human pancreatic cancer cells. Elevated levels in cells promoted EMT, migration, invasion, and metastasis in animal models, whereas RNA interference (RNAi)-mediated knockdown inhibited these processes. We determined that levels of Cav-1 and the Forkhead transcription factor FoxM1 correlated directly in pancreatic cancer cells and tumor tissues. Enforced expression of FoxM1 increased Cav-1 levels, whereas RNAi-mediated knockdown of FoxM1 had the opposite effect. FoxM1 directly bound to the promoter region of Cav-1 gene and positively transactivated its activity. Collectively, our findings defined Cav-1 as an important downstream oncogenic target of FoxM1, suggesting that dysregulated signaling of this novel FoxM1-Cav-1 pathway promotes pancreatic cancer development and progression.

KLF4-mediated negative regulation of IFITM3 expression plays a critical role in colon cancer pathogenesis.

PURPOSE: IFITM3, an IFN-inducible gene, is overexpressed in human colorectal cancer. In this study, we sought to determine the clinical significance and underlying mechanisms of its dysregulated expression in human colon tumor specimens and murine models of this disease. EXPERIMENTAL DESIGN: IFITM3 expression in a tissue microarray of tumor and matched normal colon tissue specimens and lymph node metastasis specimens obtained from 203 patients with colon cancer was measured immunohistochemically. RESULTS: IFITM3 was expressed at higher levels in colon tumors and, particularly, nodal metastases than in normal colon tissue. A Cox proportional hazards model showed that IFITM3 expression was an independent prognostic factor for disease-free survival in patients with colon cancer. Knockdown of IFITM3 expression by a specific siRNA significantly suppressed the proliferation, colony formation, migration, and invasion of colon cancer cells in vitro and tumor growth and metastasis in a xenograft model. Restored expression of KLF4, a putative tumor suppressor, downregulated IFITM3 expression in colon cancer cells in vitro. Two KLF4-binding sites in the IFITM3 promoter bound specifically to KLF4 protein in a chromatin immunoprecipitation assay and promoter mutagenesis analyses. Specific deletion of KLF4 led to IFITM3 overexpression in colon mucosa in Villin-Cre(+);Klf4(fl/fl) mice. An inverse correlation between loss of KLF4 expression and IFITM3 overexpression was evident in human colon tumors. CONCLUSION: These clinical and mechanistic findings indicate that IFITM3 is a direct transcriptional target of KLF4 and that dysregulated KLF4 expression leads to aberrant IFITM3 expression, thus contributing to colon cancer progression and metastasis.