Immune Checkpoint Blockade in Lower Gastrointestinal Cancers: A Systematic Review.

BACKGROUND: Limited therapy options exist for patients with treatment-refractory metastatic colorectal or anal cancers, prompting investigation into alternative therapies. Immunotherapy in the form of immune checkpoint blockade is one such emerging treatment that has demonstrated promising results in other tumour streams.x This review aims to assess the current use of immune checkpoint blockade in patients with lower gastrointestinal tumours. PATIENTS AND METHODS: Embase, Medline and Cochrane databases were searched for included studies. Clinical trials published in English and utilising immune checkpoint blockade for primary tumours situated in the lower gastrointestinal tract were included. Databases were searched for studies reporting on at least one of overall survival, progression-free survival or response to therapy. RESULTS: In total, 972 abstracts were screened, with 10 studies included in the final review. Eight trials (833 patients) assessed immune checkpoint blockade in the setting of colorectal cancers. These included pembrolizumab, nivolumab, durvalumab, atezolizumab, tremelimumab and ipilimumab. A total of 20 patients across all studies achieved a complete response, and 111 patients achieved a partial response to treatment. Two trials (62 patients) assessed immune checkpoint blockade in anal cancer, utilising nivolumab and pembrolizumab. Two patients across both studies achieved a complete response, and 11 patients achieved a partial response. CONCLUSIONS: A number of patients with advanced lower gastrointestinal tumours achieved a complete response to treatment for what would otherwise be considered palliative disease. Presented data have highlighted that particular patients may benefit from first-line or combination immunotherapy, and thus, further investigation is warranted to individualise treatment.

Experimental study of delivery of humidified-warm carbon dioxide during open abdominal surgery.

BACKGROUND: The aim of this study was to monitor the effect of humidified-warm carbon dioxide (HWCO2 ) delivered into the open abdomen of mice, simulating laparotomy. METHODS: Mice were anaesthetized, ventilated and subjected to an abdominal incision followed by wound retraction. In the experimental group, a diffuser device was used to deliver HWCO2 ; the control group was exposed to passive air flow. In each group of mice, surgical damage was produced on one side of the peritoneal wall. Vital signs and core temperature were monitored throughout the 1-h procedure. The peritoneum was closed and mice were allowed to recover for 24 h or 10 days. Tumour cells were delivered into half of the mice in each cohort. Tissue was then examined using scanning electron microscopy and immunohistochemistry. RESULTS: Passive air flow generated ultrastructural damage including mesothelial cell bulging/retraction and loss of microvilli, as assessed at 24 h. Evidence of surgical damage was still measurable on day 10. HWCO2 maintained normothermia, whereas open surgery alone led to hypothermia. The degree of tissue damage was significantly reduced by HWCO2 compared with that in controls. Peritoneal expression of hypoxia inducible factor 1α and vascular endothelial growth factor A was lowered by HWCO2 . These effects were also evident at the surgical damage sites, where protection from tissue trauma extended to 10 days. HWCO2 did not reduce tumorigenesis in surgically damaged sites compared with passive air flow. CONCLUSION: HWCO2 diffusion into the abdomen in the context of open surgery afforded tissue protection and accelerated tissue repair in mice, while preserving normothermia. Surgical relevance Damage to the peritoneum always occurs during open abdominal surgery, by exposure to desiccating air and by mechanical trauma/damage owing to the surgical intervention. Previous experimental studies showed that humidified-warm carbon dioxide (HWCO2 ) reduced peritoneal damage during laparoscopic insufflation. Additionally, this intervention decreased experimental peritoneal carcinomatosis compared with the use of conventional dry-cold carbon dioxide. In the present experimental study, the simple delivery of HWCO2 into the open abdomen reduced the amount of cellular damage and inflammation, and accelerated tissue repair. Sites of surgical intervention serve as ideal locations for cancer cell adhesion and subsequent tumour formation, but this was not changed measurably by the delivery of HWCO2 .

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Predicting pathological complete response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer: a systematic review.

AIM: Approximately 20% of patients treated with neoadjuvant chemoradiotherapy (nCRT) for locally advanced rectal cancer achieve a pathological complete response (pCR) while the remainder derive the benefit of improved local control and downstaging and a small proportion show a minimal response. The ability to predict which patients will benefit would allow for improved patient stratification directing therapy to those who are likely to achieve a good response, thereby avoiding ineffective treatment in those unlikely to benefit. METHOD: A systematic review of the English language literature was conducted to identify pathological factors, imaging modalities and molecular factors that predict pCR following chemoradiotherapy. PubMed, MEDLINE and Cochrane Database searches were conducted with the following keywords and MeSH search terms: 'rectal neoplasm', 'response', 'neoadjuvant', 'preoperative chemoradiation', 'tumor response'. After review of title and abstracts, 85 articles addressing the prediction of pCR were selected. RESULTS: Clear methods to predict pCR before chemoradiotherapy have not been defined. Clinical and radiological features of the primary cancer have limited ability to predict response. Molecular profiling holds the greatest potential to predict pCR but adoption of this technology will require greater concordance between cohorts for the biomarkers currently under investigation. CONCLUSION: At present no robust markers of the prediction of pCR have been identified and the topic remains an area for future research. This review critically evaluates existing literature providing an overview of the methods currently available to predict pCR to nCRT for locally advanced rectal cancer. The review also provides a comprehensive comparison of the accuracy of each modality.

New Insights on COX-2 in Chronic Inflammation Driving Breast Cancer Growth and Metastasis.

The medicinal use of aspirin stretches back to ancient times, before it was manufactured in its pure form in the late 19th century. Its accepted mechanistic target, cyclooxygenase (COX), was discovered in the 1970s and since this landmark discovery, the therapeutic application of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) has increased dramatically. The most significant benefits of NSAIDs are in conditions involving chronic inflammation (CI). Given the recognized role of CI in cancer development, the use of long-term NSAID treatment in the prevention of cancer is an enticing possibility. COX-2 is a key driver of CI, and here we review COX-2 expression as a predictor of survival in various cancer types, including breast. Obesity and post-partum involution are natural inflammatory states that are associated with increased breast cancer risk. We outline the COX-2 mediated mechanisms contributing to the growth of cancers. We dissect the cellular mechanism of epithelial-mesenchymal transition (EMT) and how COX-2 may induce this to facilitate tumor progression. Finally we examine the potential regulation of COX-2 by c-Myb, and the possible interplay between c-Myb/COX-2 in proliferation, and hypoxia inducible factor-1 alpha (HIF1α)/COX-2 in invasive pathways in breast cancer.

Selective CREB-dependent cyclin expression mediated by the PI3K and MAPK pathways supports glioma cell proliferation.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

RAD21 cohesin overexpression is a prognostic and predictive marker exacerbating poor prognosis in KRAS mutant colorectal carcinomas.

BACKGROUND: RAD21 is a component of the cohesion complex and is integral to chromosome segregation and error-free DNA repair. RAD21 is functionally important in tumour progression but its role in colorectal carcinoma (CRC) is unclear. We therefore assessed its clinicopathological and prognostic significance in CRC, as well as its effect on chemosensitivity. METHODS: A retrospective observation study examined RAD21 expression in 652 CRCs using a tissue microarray approach. Correlation with clinicopathological factors including gender, tumour grade, mucinous subtype, TNM stage, disease-specific survival (DSS), BRAF and KRAS mutation status, tumour p53 immunostaining, tumour microsatellite instability and tumour CpG island methylator phenotype was performed. Colorectal cancer cell clones with stable RAD21 knockdown were generated and tested for cellular sensitivity to conventional chemotherapeutic drugs. RESULTS: RAD21 expression was significantly correlated with male gender (56.7% vs 43.3%, P=0.02), well-differentiated histology (14.4% vs 4.0%, P=0.0001), higher T-stage (36.1% vs 27.0%, P=0.01), presence of metastasis (18.8% vs 12.6%, P=0.03), and shorter DSS (hazard ratio (HR) 1.4, 95% CI 1.1 to 1.9, P=0.01) in both univariate and multivariate analysis. RAD21 expression was associated with shorter DSS in patients with KRAS mutant tumours (HR:2.6, 95% CI:1.4-4.3, P=0.001) and in patients receiving adjuvant chemoradiotherapy (HR:1.9, 95% CI:1.2-3.0, P=0.008). Colorectal cancer cells with RAD21 knockdown exhibited enhanced sensitivity to 5-fluorouracil, either alone or in combination with oxaliplatin. CONCLUSIONS: RAD21 expression in CRC is associated with aggressive disease especially in KRAS mutant tumours and resistance to chemoradiotherapy. RAD21 may be an important novel therapeutic target.

The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis.

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.

Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling.

Aberrant Wnt signaling mediated by mutations affecting APC (adenomatous polyposis coli) or beta-catenin initiates the majority of human colorectal cancers (CRC) and drives tumorigenesis through the activation of specific genes such as MYC. We report here a novel association whereby another oncogenic transcription factor, MYB/c-Myb, is necessary for intestinal adenoma development directed by activated Wnt signaling. APC(Min/+) mice in which c-myb is haploinsufficient survive longer than wild-type APC(Min/+) animals due to a delay in adenoma formation. Intestinal adenomas from APC(Min/+) mice were assessed and found to have high levels of c-myc gene expression. We explored the relationship between activated Wnt signaling and MYB in regulating MYC and found activated beta-catenin in combination with MYB induces robust upregulation of MYC promoter activity, as well as endogenous MYC mRNA and protein expression, in human cells. This cooperation occurred through independent binding of MYB and beta-catenin to the MYC promoter. These data highlight a cooperative function for MYB in the context of activated Wnt signaling and provide a molecular basis for the expression of MYC in CRC.

Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle.

Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

Frizzled-7 dictates three-dimensional organization of colorectal cancer cell carcinoids.

Progression of colorectal cancer (CRC) involves spatial and temporal occurrences of epithelial-mesenchymal transition (EMT), whereby tumour cells acquire a more invasive and metastatic phenotype. Subsequently, the disseminated mesenchymal tumour cells must undergo a reverse transition (mesenchymal-epithelial transition, MET) at the site of metastases, as most metastases recapitulate the pathology of their corresponding primary tumours. Importantly, initiation of tumour growth at the secondary site is the rate-limiting step in metastasis. However, investigation of this dynamic reversible EMT and MET that underpins CRC morphogenesis has been hindered by a lack of suitable in vitro models. To this end, we have established a unique in vitro model of CRC morphogenesis, which we term LIM1863-Mph (morphogenetic). LIM1863-Mph cells spontaneously undergo cyclic transitions between two-dimensional monolayer (migratory, mesenchymal) and three-dimensional sphere (carcinoid, epithelial) states. Using RNAi, we demonstrate that FZD7 is necessary for MET of the monolayer cells as loss of FZD7 results in the persistence of a mesenchymal state (increased SNAI2/decreased E-cadherin). Moreover, FZD7 is also required for migration of the LIM1863-Mph monolayer cells. During development, FZD7 orchestrates either migratory or epithelialization events depending on the context. Our findings strongly implicate similar functional diversity for FZD7 during CRC morphogenesis.

Collagen loss and impaired wound healing is associated with c-Myb deficiency.

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription factor, c-Myb. When c-myb-/- embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock-out embryos fail to develop beyond day 15, fibroblasts established from these embryos (mouse embryonic fibroblasts [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c-myb-/- MEFs also exhibit slower closure than their wild-type counterparts. Embryonic lethality has meant that examination of the role of c-Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c-myb-/- MEFs, we investigated the consequences of heterozygous c-myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c-myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming growth factor-beta1 (an important collagen stimulating factor) induction expression is aberrantly high. Manipulation of c-Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders.

Transcriptional regulation of cyclo-oxygenase expression: three pillars of control.

Blocking cyclo-oxygenase (COX) isoform activities with non-steroidal anti-inflammatory drugs (NSAIDS) is widely employed in the treatment of arthritis. These agents also hold great promise in the context of pre and post-neoplastic diseases such as colorectal cancer (CRC). Nevertheless, issues of isoform specificity and delivery necessitate the exploration of other strategies to specifically block expression of the COX genes. Approaches that target gene transcription may complement enzyme inhibition. Thus, understanding the regulation of COX isoform transcription may improve the specific inhibition of expression. Three tiers of transcriptional regulation are evident: initiation, alternative splicing and messenger RNA stability. Transcription factors that activate COX-2 expression are elevated in certain disease states and emergency responses such as infection and are therefore potential targets. These factors include C/EBP-beta, phospho- CREB, NF-IL6, AP1, NFkB, and TCF-4/LEF-1. In this review we highlight another factor, c-MYB as a key COX-2 regulator in CRC. Alternative exon usage is another tier of regulation that has not received much attention. For instance, COX-1 splice variants (also known as COX-3 and PCOX-1a) may broaden the spectrum of COX activities in disease. Similarly, whilst mRNA stability is clearly modulated by steroids in the case of COX-2, the wider implications of targeting mRNA stability have not been afforded the same attention. Finally, it seems that some NSAIDS exert part of their action directly on COX-2 transcriptional regulation explaining why such agents display greater effects on this isoform than enzyme inhibition data would suggest.

c-Myb influences HIV type 1 gene expression and virus production.

c-Myb is expressed in proliferating T cells. Fifteen c-Myb-binding sites can be identified in the HIV-1 long terminal repeat (LTR), suggesting that c-Myb may regulate HIV-1 gene expression and virus replication. Increasing the cellular levels of c-Myb by transient transfection of CEM cells resulted in a 10- to 20-fold activation of HIV-1 LTR-driven gene expression and mutation of one high-affinity Myb-binding site within the LTR reduced this activation by 60 to 70%. Conversely, inhibition of c-Myb expression in MT-2 cells by treatment with c-myb antisense oligonucleotides decreased HIV-1 replication by 85%, as measured by reverse transcriptase activity and cytopathic effects. The effect of c-myb antisense oligonucleotides on HIV-1 gene expression and virus particle production appeared to be independent of cell proliferation, but dependent on the presence of c-Myb activity mediated through the HIV-1 LTR. These data show that c-myb expression affects HIV-1 replication in CD4(+) T cells.

Constitutive c-Myb amino-terminal phosphorylation and DNA binding activity uncoupled during entry and passage through the cell cycle.

The c-myb gene encodes a transcription factor that is central to hematopoietic cell growth. Phosphorylation of c-Myb by casein kinase 2 (CK2) at serines 11 and 12 has been variously implicated in the regulation of DNA binding. However, it is unclear when c-Myb phosphorylation at serines 11 and 12 occurs during the cell cycle and how this is regulated. We generated specific antisera that recognize phosphoserines 11 and 12 of c-Myb. C-Myb protein levels, extent of CK2 phosphorylation and DNA binding were then monitored following mitogenic stimulus and passage through the cell cycle in normal peripheral T-cells and the T leukemia cell line CCRF-CEM. We found that endogenous c-Myb is constitutively phosphorylated at serines 11 and 12. The amount of phosphorylated c-Myb correlates with DNA binding activity in cycling CEM cells but not upon entry of T-cells into the cell cycle. Exogenous expression of c-Myb with substitutions of serines 11 and 12 with glutamic acid or alanine had no effect on the transactivation of a c-Myb responsive reporter. These data strongly suggest that c-Myb is constitutively phosphorylated on serines 11 and 12 by CK2 or like activity and is not regulated during the cell cycle.

c-Myb and Bcl-x overexpression predicts poor prognosis in colorectal cancer: clinical and experimental findings.

The aim of this study was twofold: to assess the relationship between c-Myb and Bcl-x expression and to evaluate the prognostic significance of their expression in colorectal carcinoma (CRC) patients. Analysis of tumors from 91 CRC patients for expression of c-Myb and Bcl-x revealed a significant relationship between these two proteins. Kaplan-Meier's analysis showed an increased risk of relapse and death in patients whose tumor specimens displayed high c-Myb levels and Bcl-x positivity. Similar results were also observed excluding Dukes' D patients. Molecular analysis using three c-Myb-overexpressing LoVo clones indicated that c-Myb overexpression was accompanied by up-regulation of Bcl-x(L) protein and mRNA. Tumors originating from these clones injected in nude mice were significantly larger than those formed in mice injected with parental or vector-transfected LoVo cells. Moreover, tumors derived from parental and control vector-transfected but not from c-Myb-overexpressing LoVo cells showed high frequency of apoptotic cells. These results provide direct evidence of an association between c-Myb and Bcl-x expression and suggest that expression of both molecules might be a useful prognostic marker in CRC.

Defective stem cell factor expression in c-myb null fetal liver stroma.

High levels of c-Myb are observed in immature precursor myeloid and lymphoid cells, while downregulation of c-myb accompanies terminal differentiation to a mature phenotype. This has established c-Myb as a crucial transcription factor for hematopoiesis. Further evidence for this is the embryonic death of the c-myb homozygous mutant mouse at ED15 due to defective fetal liver erythropoiesis. Cells from fetal liver of wild-type and c-myb-/- embryos were examined in detail for their hematopoietic potential and the capacity of the stroma to support wild-type hematopoiesis. The c-myb-/- fetal liver was shown to harbor sevenfold fewer spleen focus-forming cells and a similarly lower number of cells with long-term repopulating capacity (high proliferative potential cells). However, shorter term repopulating cells were not substantially reduced. c-myb-/- stromal cells were unable to support the proliferation of wild-type bone marrow lineage-negative cells. This was found to be partly due to a decrease in stem cell factor (SCF) expression while partial rescue of the stromal cell cultures was achieved through the addition of exogenous SCF. DNA binding studies for two sites within the SCF promoter demonstrated an in vitro interaction between the SCF promoter and c-Myb and transient transfection studies demonstrated that c-Myb could substantially transactivate the SCF promoter in HEK293 cells. These data explain why the c-myb-/- embryos are so impaired in their ability to establish hematopoiesis.

Cyclooxygenase-2, a colorectal cancer nonsteroidal anti-inflammatory drug target, is regulated by c-MYB.

Cyclooxygenase-2 (COX-2) is an important pharmacological target with great promise in the prevention and treatment of colorectal cancer (CRC). The mechanism underlying COX-2 overexpression in CRC is unresolved. On the basis of the coincident high levels of the transcription factor c-MYB and COX-2 in CRC, we hypothesized that c-MYB is a candidate activator of COX-2 transcription. We identified 13 c-Myb binding sites in the human COX-2 promoter. Eight of these sites were moderate to high-affinity DNA binding targets. Promoter studies indicated that c-Myb can activate COX-2 transcription, whereas dominant-negative Myb mediated repression. These data provide the first rational basis for overexpression of COX-2 in CRC and offer an additional potential target for managing this disease.

c-Myb is critical for murine colon development.

The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.

c-Myb down-regulation is associated with human colon cell differentiation, apoptosis, and decreased Bcl-2 expression.

c-myb is expressed in human and murine colonic mucosa and elevated expression occurs in premalignant adenomatous polyps and carcinomas. c-Myb is required for colon cell proliferation, and there is evidence of c-myb down-regulation during differentiation. Recently, c-myb has been implicated in hematopoietic cell survival via regulation of bcl-2 gene expression. However, c-myb expression during terminal differentiation and apoptosis in the colonic crypt has not been examined. The experiments in this study examine the spatial and temporal expression of c-Myb protein in vivo using human colonic crypt sections and in vitro in human colon tumor cell lines undergoing butyrate-induced differentiation and apoptosis. Electron microscopy, together with molecular and biochemical analysis, was used to define the differentiation status of the cells. Results demonstrate a decrease in c-Myb expression during the commitment of cells to differentiation and apoptosis. Decreased levels of c-Myb are accompanied by a decrease in Bcl-2. These data suggest that the transcription factor c-Myb has a role in regulating the balance between proliferation, differentiation, and apoptosis in the colonic crypt. Furthermore, elevated c-Myb levels in colon tumor cells may lead to persistent bcl-2 expression, thus protecting tumor cells from programmed cell death.