Elevated translationally controlled tumour protein promotes oral cancer progression and poor outcome.

BACKGROUND: Translationally controlled tumour protein (TCTP) is a multifunctional protein elevated in multiple cancers. However, studies on its role in oral carcinogenesis and prognosis are rare. We recently reported the role of its interacting partner, MCL1, in oral cancer progression and outcome. Hence, the present study aimed to assess TCTP expression in oral tumorigenesis and its association with patient outcomes alone and in combination with MCL1. METHODS: TCTP expression was assessed by immunohistochemistry and immunoblotting in oral tissues and cells, respectively. Cell viability post siRNA/dihydroartemisinin treatment was analysed by tetrazolium salt assay. Cell survival, invasion and tumorigenic potential post TCTP knockdown were assessed by clonogenic, Matrigel and soft-agar assays, respectively. The association of TCTP with patient outcome was analysed by Kaplan-Meier and Cox regression. RESULTS: TCTP was significantly overexpressed in oral premalignant lesions (p < 0.0001), oral tumours (p < 0.0001) and oral dysplastic and cancer cells versus normal oral mucosa and also in recurrent (p < 0.05) versus non-recurrent oral tumours. Further, elevated TCTP was significantly (p < 0.05) associated with poor recurrence free survival (RFS) and poor overall survival (OS; hazard ratio = 2.29; p < 0.05). Intriguingly, the high co-expression of TCTP and MCL1 further reduced the RFS (p < 0.05) and OS (p < 0.05; hazard-ratio = 3.49; p < 0.05). Additionally, TCTP knockdown decreased survival (p < 0.05), invasion (p < 0.01) and in vitro tumorigenic potential (p < 0.0001). Dihydroartemisinin treatment reduced TCTP levels and viability of oral cancer cells. CONCLUSION: Our studies demonstrate an oncogenic role of TCTP in oral cancer progression and poor outcome. Thus, TCTP may be a potential prognostic marker and therapeutic target in oral cancers.

Plakophilin3 loss leads to an increase in autophagy and radio-resistance.

Loss of the desmosomal plaque protein plakophilin3 (PKP3) leads to increased tumor progression and metastasis. As metastatic tumors are often resistant to therapy, we wished to determine whether PKP3 loss led to increased radioresistance. PKP3 knockdown cells showed increased resistance to radiation in vitro and in vivo. The increase in resistance was accompanied by an increased ability to clear reactive oxygen species (ROS) and increased autophagy. The increase in autophagy was required for radioresistance and ROS clearance as inhibiting autophagy using either chloroquine or knocking down ATG3 re-sensitized the PKP3 knockdown clones to radiotherapy. These experiments suggest that autophagy inhibitors could target therapy-resistant PKP3 deficient tumors.

Atypical activation of signaling downstream of inactivated Bcr-Abl mediates chemoresistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) epitomises successful targeted therapy, where inhibition of tyrosine kinase activity of oncoprotein Bcr-Abl1 by imatinib, induces remission in 86% patients in initial chronic phase (CP). However, in acute phase of blast crisis, 80% patients show resistance, 40% among them despite inhibition of Bcr-Abl1 activity. This implies activation of either Bcr-Abl1- independent signalling pathways or restoration of signalling downstream of inactive Bcr-Abl1. In the present study, mass spectrometry and subsequent in silico pathway analysis of differentiators in resistant CML-CP cells identified key differentiators, 14-3-3ε and p38 MAPK, which belong to Bcr-Abl1 pathway. Their levels and activity respectively, indicated active Bcr-Abl1 pathway in CML-BC resistant cells, though Bcr-Abl1 is inhibited by imatinib. Further, contribution of these components to resistance was demonstrated by inhibition of Bcr-Abl1 down-stream signalling by knocking-out of 14-3-3ε and inhibition of p38 MAPK activity. The observations merit clinical validation to explore their translational potential.

Plakophilin3 loss leads to increased adenoma formation and rectal prolapse in APCmin mice.

Plakophilin3 (PKP3) loss leads to tumor progression and metastasis of colon cancer cells. The goal of this report was to determine if PKP3 loss led to increased disease progression in mice. We generated a colonocyte-specific knockout of PKP3 in APCmin mice, which led to increased adenoma formation, the formation of rectal prolapse, and a significant decrease in survival. The observed increase in rectal prolapse formation and decrease in survival correlated with an increase in the expression of Lipocalin2 (LCN2). Increased disease progression was observed even upon treatment with 5-fluorouracil (5FU). These results suggest that an increase in LCN2 expression might lead to therapy resistance and that LCN2 might serve as a potential therapeutic target in colorectal cancer.

Lipocalin 2 expression promotes tumor progression and therapy resistance by inhibiting ferroptosis in colorectal cancer.

Lipocalin 2 is a siderophore-binding protein that regulates iron homeostasis. Lipocalin 2 expression is elevated in multiple tumor types; however, the mechanisms that drive tumor progression upon Lipocalin 2 expression remain unclear. When Lipocalin 2 is over-expressed, it leads to resistance to 5-fluorouracil in colon cancer cell lines in vitro and in vivo by inhibiting ferroptosis. Lipocalin 2 inhibits ferroptosis by decreasing intracellular iron levels and stimulating the expression of glutathione peroxidase4 and a component of the cysteine glutamate antiporter, xCT. The increase in xCT levels is dependent on increased levels of ETS1 in Lipocalin 2 over-expressing cells. Inhibiting Lipocalin 2 function with a monoclonal antibody leads to a decrease in chemo-resistance and transformation in vitro, and a decrease in tumor progression and chemo-resistance in xenograft mouse models. Lipocalin 2 and xCT levels exhibit a positive correlation in human tumor samples suggesting that the pathway we have identified in cell lines is operative in human tumor samples. These results indicate that Lipocalin 2 is a potential therapeutic target and that the monoclonal antibody described in our study can serve as the basis for a potential therapeutic in patients who do not respond to chemotherapy.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis.

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

Centrosome Amplification and Tumorigenesis: Cause or Effect?

Centrosome amplification is a feature of multiple tumour types and has been postulated to contribute to both tumour initiation and tumour progression. This chapter focuses on the mechanisms by which an increase in centrosome number might lead to an increase or decrease in tumour progression and the role of proteins that regulate centrosome number in driving tumorigenesis.

14-3-3 proteins mediate the localization of Centrin2 to centrosome.

14-3-3ε and 14-3-3γ localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As 14-3-3γ and 14-3-3ε form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosome duplication. The results in this report demonstrate that 14-3-3ε and 14-3-3γ form a complex with Centrin2 and that the binding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with 14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggest that in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also be required for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.

Plakophilin3 loss leads to an increase in lipocalin2 expression, which is required for tumour formation.

An increase in tumour formation and metastasis are observed upon plakophilin3 (PKP3) loss. To identify pathways downstream of PKP3 loss that are required for increased tumour formation, a gene expression analysis was performed, which demonstrated that the expression of lipocalin2 (LCN2) was elevated upon PKP3 loss and this is consistent with expression data from human tumour samples suggesting that PKP3 loss correlates with an increase in LCN2 expression. PKP3 loss leads to an increase in invasion, tumour formation and metastasis and these phenotypes were dependent on the increase in LCN2 expression. The increased LCN2 expression was due to an increase in the activation of p38 MAPK in the HCT116 derived PKP3 knockdown clones as LCN2 expression decreased upon inhibition of p38 MAPK. The phosphorylated active form of p38 MAPK is translocated to the nucleus upon PKP3 loss and is dependent on complex formation between p38 MAPK and PKP3. WT PKP3 inhibits LCN2 reporter activity in PKP3 knockdown cells but a PKP3 mutant that fails to form a complex with p38 MAPK cannot suppress LCN2 promoter activity. Further, LCN2 expression is decreased upon loss of p38ß, but not p38α, in the PKP3 knockdown cells. These results suggest that PKP3 loss leads to an increase in the nuclear translocation of p38 MAPK and p38ß MAPK is required for the increase in LCN2 expression.

Plakoglobin localization to the cell border restores desmosome function in cells lacking 14-3-3γ.

Desmosomes are cell-cell adhesion junctions that anchor intermediate filaments. Loss of 14-3-3γ in HCT116 cells led to defects in desmosome assembly due to a decrease in the transport of Plakoglobin (PG) to the cell border thus disrupting desmosome formation. Desmosome formation in cells lacking 14-3-3γ was restored by artificially localizing PG to the cell border by fusing it to EGFP-f (PG-EGFP-f). These results suggest that a major role of 14-3-3γ in desmosome assembly is to transport PG to the cell border leading to the initiation of desmosome formation.

Plakophilin3 increases desmosome assembly, size and stability by increasing expression of desmocollin2.

Previous reports show that the desmosomal plaque protein plakophilin3 (PKP3) is essential for desmosome formation. Here, we report that PKP3 over-expression decreases calcium dependency for de novo desmosome formation and makes existing cell-cell adhesion junctions more resilient in low calcium medium due to an increase in desmocollin2 expression. PKP3 overexpression increases the stability of other desmosomal proteins independently of the increase in DSC2 levels and regulates desmosome formation and stability by a multimodal mechanism affecting transcription, protein stability and cell border localization of desmosomal proteins.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.

MMP7 is required to mediate cell invasion and tumor formation upon Plakophilin3 loss.

Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation.

Generation of transgenic mice by exploiting spermatogonial stem cells in vivo.

The protocols in this chapter describe two techniques for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells (SSCs) with a high rate of success. SSCs in prepubescent animals can either be infected in vivo with recombinant lentiviruses expressing the transgene of interest or DNA can be injected into the testis followed by the application of an electric current resulting in integration of the linearized DNA containing a transgene downstream of the appropriate promoter into SSCs. All male pre-founder mice produced transgenic pups using both protocols with the transgene being heritable. Further, the pre-founder mice could be used in multiple mating experiments resulting in the generation of multiple progeny. These protocols could be extended to perform over-expression/knockdown screens in vivo using bar-coded lentiviruses/plasmid constructs, thus permitting the design of genetic screens in the mouse. Further, these protocols could be adapted to achieve transgenesis in other laboratory animals resulting in the generation of model systems that closely approximate human development and disease.

14-3-3γ-Mediated transport of plakoglobin to the cell border is required for the initiation of desmosome assembly in vitro and in vivo.

The regulation of cell-cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3γ leads to a decrease in cell-cell adhesion and a defect in the transport of plakoglobin and other desmosomal proteins to the cell border in HCT116 cells and cells of the mouse testis. 14-3-3γ binds to plakoglobin in a PKCµ-dependent fashion, resulting in microtubule-dependent transport of plakoglobin to cell borders. Transport of plakoglobin to the border is dependent on the KIF5B-KLC1 complex. Knockdown of KIF5B in HCT116 cells, or in the mouse testis, results in a phenotype similar to that observed upon 14-3-3γ knockdown. Our results suggest that loss of 14-3-3γ leads to decreased desmosome formation and a decrease in cell-cell adhesion in vitro, and in the mouse testis in vivo, leading to defects in testis organization and spermatogenesis.

Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

BACKGROUND: Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. CONCLUSIONS/SIGNIFICANCE: These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.