Essential Role of Cdc42 in Ras-Induced Transformation Revealed by Gene Targeting

The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies

MTG16 regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors

Aberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (iBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium-induced (DSS-induced) colitis. Transcriptomic analyses implicated increased Ephrussi box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16(P20ST)), we showed that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared with unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16(-/)(-) colon in the azoxymethane/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets. is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.Peer reviewe

Growth defects observed in HRasV12-transformed cells following Cdc42 loss are partially rescued by activation of Akt signaling.

<p>(<b>a</b>) Cell lysates from non-transformed and HRasV12-transformed cells were immunoblotted for phospho-Akt. Gapdh serves as a loading control. (<b>b</b>) Cells were infected with Ad-GFP, Ad-GFP-Cre or Ad-GFP-Cre + Ad-myr-Akt. Cell lysates were obtained and blotted for Akt expression. (<b>c</b>) Exponentially growing cells were pulsed with BrdU for 1 hour prior to harvest. Cells were stained with anti-BrdU and PI and analyzed by flow cytometry. (<b>d</b>) PI staining was used to determine the percentage of cells with 2N DNA content. Graphs represent at least three independent experiments.</p

Cdc42 loss does not impair transformation by the oncoprotein, c-Myc.

<p>(<b>a</b>) p53-immortalized cells were infected with a retrovirus expressing the oncoprotein, c-Myc. c-Myc overexpression was verified by western blot analysis. (<b>b</b>) Pull-down assays were performed with GST-PAK1 to determine the relative abundance of activated Cdc42 in HRasV12 vs. c-Myc transformed cells. (<b>c</b>) Lysates from c-Myc transformed cells infected with Ad-GFP or Ad-GFP-Cre were immunoblotted for Cdc42. (<b>d</b>) Cultured c-Myc transformed cells were visualized 4 days following <i>cdc42</i> deletion by bright field microscopy (upper 2 panels). Cells were fixed and stained with rhodamine-phalloidin and DAPI to visualize actin structures (lower 2 panels). (<b>e</b>) An equal number of c-Myc transformed Cdc42 proficient and deficient cells were seeded. Cells were counted every three days by trypan blue exclusion. Curves represent at least two independent experiments performed in triplicate. (<b>f</b>) Cdc42-proficient and –deficient c-Myc cells were grown in a 0.3% agarose solution. Experiments were performed in triplicate and 9 independent fields were counted for colonies. ns = not significant.</p

Cdc42 activity is increased upon Ras-mediated transformation.

<p>(<b>a</b>) Cell immortalization was achieved following retroviral transduction of cdc42^f/f cells with a dominant-negative p53 (p53dd). Dominant-negative expression was determined by western blot for p53. (<b>b</b>) Immortalized, cdc42^f/f cells were transformed through retroviral transduction with HRasV12. Transformed cells exhibit HRas overexpression and increased active, GTP-bound HRas as determined by pull-down with the ras binding domain (RBD) of Raf1 fused to GST. (<b>c</b>) HRasV12 transformed cells exhibit a significant increase in Cdc42 activity compared to non-transformed control cells. Pull-down assays were performed with GST-PAK1. The graphed data represents densitometry quantification from three independent experiments.</p

Cdc42-deficiency impairs HRasV12-driven tumorigenesis.

<p>(<b>a</b>) Cdc42 proficient and deficient cells in a 1∶1 PBS:matrigel mixture were subcutaneously injected into the flanks of immunocompromised nude mice. Developing tumors were measured at indicated time points with calipers to determine tumor volume. n = 6. (<b>b</b>) 16 days post-injection mice were sacrificed and tumors excised and weighed (upper panel). A representative image of tumors from three mice are shown (lower panel). (<b>c</b>) Tumors were fixed, sectioned and stained with hematoxylin and eosin. (<b>d</b>) Left panel - The recombination status of the <i>cdc42</i> locus was determined for cells prior to injection into nude mice by genomic PCR. Right panel - DNA was isolated from resulting subcutaneous tumors and the recombination status of <i>cdc42</i> determined by PCR. Tumors from two representative mice were shown. The upper band corresponds to the non-recombined loxp/loxp allele from injected cells, the middle band corresponds to the wild-type <i>cdc42</i> allele derived from stromal cells derived from the recipient mouse, and the lower band corresponds to the recombined <i>cdc42</i> allele.</p

Loss of Cdc42 in HRasV12 transformed cells results in reduced cell proliferation and anchorage-independent growth.

<p>(<b>a</b>) Equal cell numbers were plated following cdc42 deletion and cells were counted by trypan blue exclusion every three days for 9 days. Curves represent at least two independent experiments performed in triplicate. (<b>b</b>) Cdc42-proficient and –deficient HRasV12 cells were grown in a 0.3% agarose solution. Experiments were performed in triplicate and 9 independent fields were counted for colonies. (<b>c</b>) Exponentially growing cells were pulsed with 5-bromodeoxyuridine (BrdU) for 1 hr. prior to harvest. Cells were stained with anti-BrdU and propidium iodide (PI) and analyzed by flow cytometry. Graphs represent at least three independent experiments. (<b>d</b>) Cell lysates were immunoblotted for the cell cycle regulators, Cyclin D1 and p16^ink4a. Gapdh served as a loading control.</p

A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets

Summary: Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.For complete details on the use and execution of this protocol, please refer to Stengel et al., 2021)