Epidemiological Characteristics of Cancer Patients Attending at Felege Hiwot Referral Hospital, Northwest Ethiopia.

BACKGROUND: Cancer has become a public health problem and a challenge in developing countries, including Ethiopia. There is scanty local data on cancer epidemiology in Amhara region, Ethiopia. Thus, this study aimed to describe epidemiological characteristics of cancer patients attending Felege Hiwot Referral Hospital. METHODS: This study was based on a patient cancer registry that took place in Bahir Dar Felege Hiwot Referral Hospital, Amhara Regional State, Ethiopia. It is the main referral hospital in the Amhara region, and serves more than 5 million people. The hospital has units including oncology for follow-up health care services. All confirmed cancer patients attending oncology units from July 2017 to June 2019 were included in the study. Global Moran's I statistic was employed to assess spatial heterogeneity of cancer cases across districts. Getis-Ord Gi* statistics was performed to identify hot spot districts with high numbers of cancer cases. RESULTS: In a two-year period, a total of 1888 confirmed cancer patients were registered. There was a significant variation of cancer patients between females (60.8% 95%CI 58.5 to 63.0%) and males (39.3% 95%CI 37.0 to 41.5%). The first three most frequent cancer types seen were breast (19.4%) and cervical cancer (12.9%), and lymphoma (15.7%). Breast and cervical cancer and lymphoma were the first three cancers type among women, whereas lymphoma, sarcoma, and lung cancer were the three most common cancer among men. Spatially, cancer cases were non-random in the study area (global Moran's I = 0.25, z-score = 5.6, p-value < 0.001). Bahir Dar city administration (z = 3.93, p < 0.001), Mecha (z = 3.49, p < 0.001), Adet (z = 3.25, p < 0.01), Achefer (z = 3.29, p < 0.001), Dangila (z = 3.32, p < 0.001), Fogera (z = 2.19, p < 0.05), and Dera (z = 2.97, p < 0.01) were spatially clustered as hotspot with high numbers of cluster cases. CONCLUSIONS: We found that there is a variation in the cancer types with sex. This study provides an insight for further exploration of environmental and occupational exposure related factors for cancer to guide future cancer prevention and control programs. The current study also calls for expansion of cancer registry sites, including in rural areas in the region.

KLF4 Regulates Metabolic Homeostasis in Response to Stress.

Cancerous cells are detrimental to the human body and can be incredibly resilient against treatments because of the complexities of molecular carcinogenic pathways. In particular, cancer cells are able to sustain increased growth under metabolic stress due to phenomena like the Warburg effect. Krüppel-like factor 4 (KLF4), a context-dependent transcription factor that can act as both a tumor suppressor and an oncogene, is involved in many molecular pathways that respond to low glucose and increased reactive oxygen species (ROS), raising the question of its role in metabolic stress as a result of increased proliferation of tumor cells. In this study, metabolic assays were performed, showing enhanced efficiency of energy production in cells expressing KLF4. Western blotting showed that KLF4 increases the expression of essential glycolytic proteins. Furthermore, we used immunostaining to show that KLF4 increases the localization of glucose transporter 1 (GLUT1) to the cellular membrane. 2',7'-Dichlorodihydrofluorescein diacetate (H2DCF-DA) was used to analyze the production of ROS, and we found that KLF4 reduces stress-induced ROS within cells. Finally, we demonstrated increased autophagic death in KLF4-expressing cells in response to glucose starvation. Collectively, these results relate KLF4 to non-Warburg metabolic behaviors that support its role as a tumor suppressor and could make KLF4 a target for new cancer treatments.

Cells deficient for Krüppel-like factor 4 exhibit mitochondrial dysfunction and impaired mitophagy.

Krüppel-like factor 4 (Human Protein: KLF4; Human Gene: Klf4; Murine Protein: KLF4; Murine Gene: Klf4) is a zinc finger-containing transcription factor with diverse regulatory functions. Mouse embryonic fibroblasts (MEFs) lacking Klf4 exhibit genomic instability, increased reactive oxygen species (ROS), and decreased autophagy. Elevated ROS is linked to impairments in mitochondrial damage recovery responses and is often tied to disruption in mitochondrial-targeted autophagy known as mitophagy. In this study, we sought to identify a mechanistic connection between KLF4 and mitophagy. Using flow cytometry, we found that Klf4-null MEFs have diminished ability to recover mitochondrial health and regulate ROS levels after mitochondrial damage. Confocal microscopy indicated decreased localization of autophagy protein LC3 to mitochondria following mitochondrial damage in Klf4-null cells, suggesting decreased mitophagy. Western blotting and RT-PCR revealed decreased mRNA and protein expression of the mitophagy-associated protein Bnip3 and antioxidant protein GSTα4 in Klf4-null cells, providing a rationale for their impaired mitophagy and ROS accumulation. Inducing Bnip3 expression in these cells recovered mitophagy but did not decrease ROS accumulation. Our findings suggest that in Klf4-null cells, decreased Bnip3 expression impairs mitophagy and is associated with increased mitochondrial ROS production after mitochondrial damage, providing a rationale for their genomic instability and supports a tumor suppressive role for KLF4 in certain tumors as previously observed.

Krüppel-like factor 4 mediates cellular migration and invasion by altering RhoA activity.

Kru¨ppel like factor 4 (KLF4) is a transcription factor that regulates genes related to differentiation and proliferation. KLF4 also plays a role in metastasis via epithelial to mesenchymal transition. Here, we investigate the function of Klf4 in migration and invasion using mouse embryonic fibroblasts and the RKO human colon cancer cell line. Compared to wild-type, cells lacking Klf4 exhibited increased migration-associated phenotypes. In addition, overexpression of Klf4 in Klf4-/- MEFs attenuated the presence of stress fibers to wild-type levels. An invasion assay suggested that lack of Klf4 resulted in increased invasive capacity. Finally, analysis of RhoA showed elevated RhoA activity in both RKO and MEF cells. Taken together, our results strongly support the novel role of KLF4 in a post-translational regulatory mechanism where KLF4 indirectly modulates the actin cytoskeleton morphology via activity of RhoA in order to inhibit cellular migration and invasion.

Impaired autophagy in mouse embryonic fibroblasts null for Krüppel-like Factor 4 promotes DNA damage and increases apoptosis upon serum starvation.

BACKGROUND: Autophagy is a major cellular process by which cytoplasmic components such as damaged organelles and misfolded proteins are recycled. Although low levels of autophagy occur in cells under basal conditions, certain cellular stresses including nutrient depletion, DNA damage, and oxidative stress are known to robustly induce autophagy. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor activated during oxidative stress to maintain genomic stability. Both autophagy and KLF4 play important roles in response to stress and function in tumor suppression. METHODS: To explore the role of KLF4 on autophagy in mouse embryonic fibroblasts (MEFs), we compared wild-type with Klf4 deficient cells. To determine the levels of autophagy, we starved MEFs for different times with Earle's balanced salts solution (EBSS). Rapamycin was used to manipulate mTOR activity and autophagy. The percentage of cells with γ-H2AX foci, a marker for DNA damage, and punctate pattern of GFP-LC3 were counted by confocal microscopy. The effects of the drug treatments, Klf4 overexpression, or Klf4 transient silencing on autophagy were analyzed using Western blot. Trypan Blue assay and flow cytometry were used to study cell viability and apoptosis, respectively. qPCR was also used to assay basal and the effects of Klf4 overexpression on Atg7 expression levels. RESULTS: Here our data suggested that Klf4 (-/-) MEFs exhibited impaired autophagy, which sensitized them to cell death under nutrient deprivation. Secondly, DNA damage in Klf4-null MEFs increased after treatment with EBSS and was correlated with increased apoptosis. Thirdly, we found that Klf4 (-/-) MEFs showed hyperactive mTOR activity. Furthermore, we demonstrated that rapamycin reduced the increased level of mTOR in Klf4 (-/-) MEFs, but did not restore the level of autophagy. Finally, re-expression of Klf4 in Klf4 deficient MEFs resulted in increased levels of LC3II, a marker for autophagy, and Atg7 expression level when compared to GFP-control transfected Klf4 (-/-) MEFs. CONCLUSION: Taken together, our results strongly suggest that KLF4 plays a critical role in the regulation of autophagy and suppression of mTOR activity. In addition, we showed that rapamycin decreased the level of mTOR in Klf4 (-/-) MEFs, but did not restore autophagy. This suggests that KLF4 regulates autophagy through both mTOR-dependent and independent mechanisms. Furthermore, for the first time, our findings provide novel insights into the mechanism by which KLF4 perhaps prevents DNA damage and apoptosis through activation of autophagy.

Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for Krüppel-like factor 4.

Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-ß-Gal staining, we show that Klf4(-/-) MEFs enter senescence earlier than Klf4(+/+) MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against γ-H2AX indicates that the increased level of DNA damage in Klf4(-/-) MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4(-/-) MEFs, treatment with NAC, reduces the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4(-/-) MEFs have decreased expression of the antioxidant gene, Gsta4. The downregulation of the Gsta4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes.

Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts.

BACKGROUND: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy. METHODS: To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs null for Klf4 (Klf4(-/-)), we transfected Klf4(-/-)MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4(+/+)) and untransfected or mock-transfected Klf4(-/-)MEFs. RESULTS: We show that overexpression of Klf4 in Klf4(-/-)MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation. CONCLUSION: Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

The E3 ubiquitin ligase SMAD ubiquitination regulatory factor 2 negatively regulates Krüppel-like factor 5 protein.

The zinc finger transcription factor Krüppel-like factor 5 (KLF5) is regulated posttranslationally. We identified SMAD ubiquitination regulatory factor 2 (SMURF2), an E3 ubiquitin ligase, as an interacting protein of KLF5 by yeast two-hybrid screen, coimmunoprecipitation, and indirect immunofluorescence studies. The SMURF2-interacting domains in KLF5 were mapped to its carboxyl terminus, including the PY motif of KLF5 and its zinc finger DNA-binding domain. KLF5 protein levels were reduced significantly upon overexpression of SMURF2 but not catalytically inactive SMURF2-C716A mutant or SMURF1. SMURF2 alone reduced the protein stability of KLF5 as shown by cycloheximide chase assay, indicating that SMURF2 specifically destabilizes KLF5. In contrast, KLF5(1-165), a KLF5 amino-terminal construct that lacks the PY motif and DNA binding domain, was not degraded by SMURF2. The degradation of KLF5 by SMURF2 was blocked by the proteasome inhibitor MG132, and SMURF2 efficiently ubiquitinated both overexpressed and endogenous KLF5. In contrast, knocking down SMURF2 by siRNAs significantly enhanced KLF5 protein levels, reduced ubiquitination of KLF5, and increased the expression of cyclin D1 and PDGF-A, two established KLF5 target genes. In consistence, SMURF2, but not the E3 ligase mutant SMURF2-C716A, significantly inhibited the transcriptional activity of KLF5, as demonstrated by dual luciferase assay using the PDGF-A promoter, and suppressed the ability of KLF5 to stimulate cell proliferation as measured by BrdU incorporation. Hence, SMURF2 is a novel E3 ubiquitin ligase for KLF5 and negatively regulates KLF5 by targeting it for proteasomal degradation.

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts.

BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. METHODS: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. RESULTS: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. CONCLUSIONS: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.

Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish.

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce their own expression through a positive feedback loop. We show that two tissue specific enhancers in the zebrafish squint gene mediate the response to Nodal signals. Expression in the blastomeres depends upon a conserved Nodal response element (NRE) in the squint first intron, while expression in the extra-embryonic enveloping layer (EVL) is mediated by an element upstream of the transcription start site. Targeted depletion experiments demonstrate that the zebrafish Nodal-related proteins Squint and Cyclops are required in the YSL for endoderm and head mesoderm formation. Thus, Nodal signals mediate interactions between embryonic and extra-embryonic tissues in zebrafish that maintain nodal-related gene expression in the margin. Our results demonstrate a high degree of functional conservation between the extra-embryonic tissues of mouse and zebrafish.

The role of maternal Activin-like signals in zebrafish embryos.

Maternal Activin-like proteins, a subgroup of the TGF-beta superfamily, play a key role in establishing the body axes in many vertebrates, but their role in teleosts is unclear. At least two maternal Activin-like proteins are expressed in zebrafish, including the Vg1 orthologue, zDVR-1, and the nodal-related gene, Squint. Our analysis of embryos lacking both maternal and zygotic squint function revealed that maternal squint is required in some genetic backgrounds for the formation of dorsal and anterior tissues. Conditional inactivation of the ALK4, 5 and 7 receptors by SB-505124 treatment during the cleavage stages ruled out a role for maternal Squint, zDVR-1, or other Activin-like ligands before the mid-blastula transition, when the dorsal axis is established. Furthermore, we show that maternal Squint and zDVR-1 are not required during the cleavage stages to induce zygotic nodal-related gene expression. nodal-related gene expression decreases when receptor inhibition continues past the mid-blastula transition, resulting in a progressive loss of mesoderm and endoderm. We conclude that maternally expressed Activin-like signals do not act before the mid-blastula transition in zebrafish, but do have a variably penetrant role in the later stages of axis formation. This contrasts with the early role for these signals during Xenopus development.

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish.

BACKGROUND: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-beta superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects. RESULTS: We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated. CONCLUSION: We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.