Murali R. Kuracha

Histone posttranslational modifications and cell fate determination: lens induction requires the lysine acetyltransferases CBP and p300

Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300−/− ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.

Spry1 and Spry2 are necessary for lens vesicle separation and corneal differentiation

PURPOSE The studies reported here were performed to analyze the roles of Sproutys (Sprys), downstream targets and negative feedback regulators of the fibroblast growth factor (FGF) signaling pathway, in lens and corneal differentiation. METHODS Spry1 and -2 were conditionally deleted in the lens and corneal epithelial precursors using the Le-Cre transgene and floxed alleles of Spry1 and -2. Alterations in lens and corneal development were assessed by hematoxylin and eosin staining, in situ hybridization, and immunohistochemistry. RESULTS Spry1 and -2 were upregulated in the lens fibers at the onset of fiber differentiation. FGF signaling was both necessary and sufficient for induction of Spry1 and -2 in the lens fiber cells. Spry1 and -2 single- or double-null lenses failed to separate from the overlying ectoderm and showed persistent keratolenticular stalks. Apoptosis of stalk cells, normally seen during lens vesicle detachment from the ectoderm, was inhibited in Spry mutant lenses, with concomitant ERK activation. Prox1 and p57(KIP2), normally upregulated at the onset of fiber differentiation were prematurely induced in the Spry mutant lens epithelial cells. However, terminal differentiation markers such as β- or γ-crystallin were not induced. Corneal epithelial precursors in Spry1 and -2 double mutants showed increased proliferation with elevated expression of Erm and DUSP6 and decreased expression of the corneal differentiation marker K12. CONCLUSIONS Collectively, the results indicate that Spry1 and -2 (1) through negative modulation of ERKs allow lens vesicle separation, (2) are targets of FGF signaling in the lens during initiation of fiber differentiation and (3) function redundantly in the corneal epithelial cells to suppress proliferation.

Activated Ras alters lens and corneal development through induction of distinct downstream targets

BackgroundMammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF) signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas.ResultsRas transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas.ConclusionsCollectively, these results suggest that Ras activation a) induces distinct sets of downstream targets in the lens and cornea resulting in distinct cellular responses and b) is sufficient for initiation but not completion of lens fiber differentiation.

Novel homozygous, heterozygous and hemizygous FRMD7 gene mutations segregated in the same consanguineous family with congenital X-linked nystagmus

Congenital nystagmus (NYS) is characterized by bilateral, spontaneous, and involuntary movements of the eyeballs that most commonly presents between 2 and 6 months of life. To date, 44 different FRMD7 gene mutations have been found to be etiological factors for the NYS1 locus at Xq26-q27. The aim of this study was to find the FRMD7 gene mutations in a large eleven-generation Indian pedigree with 71 members who are affected by NYS. Mutation analysis of the entire coding region and splice junctions of the FRMD7 gene revealed a novel missense mutation, c.A917G, predicts a substitution of Arg for Gln at codon 305 (Q305R) within exon 10 of FRMD7. The mutation was detected in hemizygous males, and in homozygous and heterozygous states in affected female members of the family. This mutation was not detected in unaffected members of the family or in 100 unrelated control subjects. This mutation was found to be at a highly conserved residue within the FERM-adjacent domain in affected members of the family. Structure prediction and energetic analysis of wild-type FRMD7 compared with mutant (Q305R) revealed that this change in amino acid led to a change in secondary structure predicted to be an energetically unstable protein. The present study represents the first confirmation of FRMD7 gene mutations in a multigenerational Indian family and expands the mutation spectrum for this locus.

Expanding the spectrum of γ-secretase gene mutation-associated phenotypes: two novel mutations segregating with familial hidradenitis suppurativa (acne inversa) and acne conglobata.

phenotypes: two novel mutations segregating with familial hidradenitis suppurativa (acne inversa) and acne conglobata Uppala Ratnamala, Devendrasinh Jhala, Nayan K. Jain, Nazia M. Saiyed, Meda Raveendrababu, Mandava V. Rao, Timir Y. Mehta, Faiza M. Al-Ali, Kavi Raval, Sreelatha Nair, Nair K. Chandramohan, Murali R. Kuracha, Swapan K. Nath and Uppala Radhakrishna Department of Pharmacology, Creighton University, Omaha, NE, USA; Department of Life Sciences, Gujarat University, Ahmedabad, India; Department of Zoology, School of Science, Gujarat University, Ahmedabad, India; Department of Biotechnology, Institute of Science, Nirma University, Ahmedabad, India; Samarpan Medical & Research Organization on Skin, Modasa, India; Dermatology Centre, Rashid Hospital, Dubai Health Authority, Dubai, UAE; Department of Radiology, Sinai-Grace Hospital, Detroit, MI, USA; Department of Fetal Medicine, Lifeline Genetics and Research Centre, Lifeline Hospital, Adoor, India; Division of Surgical Oncology, Regional Cancer Centre, Thiruvananthapuram, India; Cancer Center, Creighton University, Omaha, NE, USA; Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Green Cross Voluntary Blood Bank, Ahmedabad, India; Obstetrics and Gynecology, Oakland University William Beaumont School of Medicine, Royal Oak, MI, USA Correspondence: Uppala Radhakrishna, PhD, Obstetrics and Gynecology, Oakland University William Beaumont School of Medicine, Royal Oak, MI, USA, Tel.: +248 551 2574, Fax: 2485512947, e-mail: Uppala.Radhakrishna@beaumont.edu

Patient-derived xenograft mouse models of pseudomyxoma peritonei recapitulate the human inflammatory tumor microenvironment

Pseudomyxoma peritonei (PMP) is a neoplastic syndrome characterized by peritoneal tumor implants with copious mucinous ascites. The standard of care for PMP patients is aggressive cytoreductive surgery performed in conjunction with heated intraperitoneal chemotherapy. Not all patients are candidates for these procedures and a majority of the patients will have recurrent disease. In addition to secreted mucin, inflammation and fibrosis are central to PMP pathogenesis but the molecular processes that regulate tumor‐stromal interactions within the peritoneal tumor microenvironment remain largely unknown. This knowledge is critical not only to elucidate PMP pathobiology but also to identify novel targets for therapy. Here, we report the generation of patient‐derived xenograft (PDX) mouse models for PMP and assess the ability of these models to replicate the inflammatory peritoneal microenvironment of human PMP patients. PDX mouse models of low‐ and high‐grade PMP were generated and were of a similar histopathology as human PMP. Cytokines previously shown to be elevated in human PMP were also elevated in PDX ascites. Significant differences in IL‐6 and IL‐8/KC/MIP2 were seen between human and PDX ascites. Interestingly, these cytokines were mostly secreted by mouse‐derived, tumor‐associated stromal cells rather than by human‐derived PMP tumor cells. Our data suggest that the PMP PDX mouse models are especially suited to the study of tumor‐stromal interactions that regulate the peritoneal inflammatory environment in PMP as the tumor and stromal cells in these mouse models are of human and murine origins, respectively. These mouse models are therefore, likely to be useful in vivo surrogates for testing and developing novel therapeutic treatment interventions for PMP.

The endocytic recycling regulatory protein EHD1 Is required for ocular lens development

The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis.

Bilateral blockade of MEK- and PI3K-mediated pathways downstream of mutant KRAS as a treatment approach for peritoneal mucinous malignancies

Mucinous colorectal adenocarcinomas (MCAs) are clinically and morphologically distinct from nonmucinous colorectal cancers (CRCs), show a distinct spectrum of genetic alterations (higher KRAS mutations, lower p53, high MUC2), exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement) and are associated with poorer response to chemotherapy with limited treatment options. Here, we report the effectiveness of combinatorial targeting of two KRAS-mediated parallel pathways in reducing MUC2 production and mucinous tumor growth in vitro and in vivo. By knockdown of mutant KRAS we show that, mutant KRAS (a) is necessary for MUC2 production in vitro and (b) synergistically engages PI3K/AKT and MEK/ERK pathways to maintain MUC2 expression in MCA cells. These results define a novel and a previously undescribed role for oncogenic KRAS in mucinous cancers. MCA cells were sensitive to MEK inhibition suggesting cellular dependence (‘addiction’) of KRAS-mutant MCA cells on hyperactivation of the MEK-driven pathway. Interestingly, MCA cells, though initially sensitive, were later resistant to PI3K single agent inhibition. Our studies suggest that this resistance involves dynamic rewiring of signaling circuits mediated through relief of RTK inhibition and MEK-ERK rebound activation. This resistance however, could be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEK- and PI3K-targeted combination therapy for not only KRAS mutant MCA but also for other related mucinous neoplasms that overproduce MUC2 and have a high rate of KRAS mutations such as pseudomyxoma peritonei.

Abstract 2661: Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells

Purpose. A significant proportion of sporadic colorectal cancers (CRCs) (10-15%) are of the mucinous subtype (>50% of the tumor). Mucinous colorectal adenocarcinomas (MCAs) are clinically, morphologically and molecularly distinct from nonmucinous CRCs as they show a different spectrum of genetic alterations (higher KRAS mutations, MSI-H, lower p53, high MUC2) and exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement). MCAs are associated with poorer response to chemotherapy and have limited treatment options. The purpose of this study was to test the effectiveness of combinatorial targeting of two KRAS-associated parallel pathways in an attempt to counter adaptive resistance associated with single pathway inhibition. Methods. Two KRAS mutant MCA cell lines LS174T and RW7213 were treated with two small molecule inhibitors, GDC0973 and GDC0941, to block mitogen-activated extracellular regulated kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) respectively. Adaptive resistance of these two cell lines to the PI3K inhibitor (PI3Ki) was examined by protein phospho-receptor tyrosine kinase (RTK) arrays, western blots and immunocytochemistry. Results. Bioinformatic analysis of the colon cancer datasets in the cancer genome atlas (TCGA) showed increased mutation rates in mucinous over nonmucinous CRCs for effectors of RAS-RAF-MEK-ERK (80% versus 41.9%) and PI3K-AKT-mTOR (60% versus 21.8%) pathways. Both MCA cell lines were sensitive to MEK inhibitor (MEKi) treatment. In contrast, these cell lines, though initially sensitive to PI3Ki, later became resistant. Concomitant with the development of resistance were a) increase in phosphorylated RTKs including IR, IGF-1R, EGFR, ErbB2 b) increase in phosphorylated ERK and AKT, c) nuclear translocation of the transcription factor FOXO3A and d) a decrease in proapoptotic protein BIM. Combinatorial treatment of PI3Ki and MEKi synergistically reduced viability of MCA tumor cells with attendant decrease in phosphorylated ERK and AKT and increase in BIM. Conclusions. Our data suggest cellular dependence (‘addiction’) of KRAS-mutant mucinous CRC cells on hyperactivation of MEK and PI3K pathways. Interestingly, PI3K single agent inhibition initially triggers pathway downregulation and reduced tumor cell viability but later leads to development of adaptive resistance. Our studies suggest this resistance involves dynamic rewiring of signaling circuits mediated through FOXO3A nuclear localization, relief of RTK inhibition, MEK-ERK activation and BIM reduction. Adaptive resistance however, can be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEKi and PI3Ki combination therapy for KRAS mutant CRCs. Citation Format: Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2015-2661