PDF file - 118KB, Supplementary Figure S1 shows gating strategies for flow cytometry, quantitatio... more PDF file - 118KB, Supplementary Figure S1 shows gating strategies for flow cytometry, quantitation for EpCAM expression level and EpCAM+ cells initiated tumors in mice.
PDF file - 20KB, Supplementary Table S4 shows p53 mutation status and proportion of CD44+ cells i... more PDF file - 20KB, Supplementary Table S4 shows p53 mutation status and proportion of CD44+ cells in gastric tumor xenografts.
PDF file - 24KB, Supplementary Table S3 shows effects of CD44 variant over-expression and knockdo... more PDF file - 24KB, Supplementary Table S3 shows effects of CD44 variant over-expression and knockdown on tumor initiation potential.
PDF file - 93KB, Supplementary Figure S4 shows gating strategy for sorting of CD44 depleted TMK1 ... more PDF file - 93KB, Supplementary Figure S4 shows gating strategy for sorting of CD44 depleted TMK1 cells for tumor initiation assay and xenograft tumors formed.
PDF file - 130KB, Supplementary Figure S3 shows fractionation of CD44v+ cells for limiting diluti... more PDF file - 130KB, Supplementary Figure S3 shows fractionation of CD44v+ cells for limiting dilution analysis in vitro and in vivo.
PDF file - 25KB, Supplementary Table S2 shows estimation of CSC frequency in unsorted, EpCAM+CD13... more PDF file - 25KB, Supplementary Table S2 shows estimation of CSC frequency in unsorted, EpCAM+CD133+ and EpCAM+CD133- fractions from GC16 xenografts.
The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (... more The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define ...
The surfacemarker CD44 has been identified as one of several markers associated with cancer stem ... more The surfacemarker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues.We identified CD44v8-10 as the predominantCD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Recipro...
Histone modifications play diverse and essential roles in the maintenance of genome integrity, as... more Histone modifications play diverse and essential roles in the maintenance of genome integrity, as evidenced by the frequency at which different histone marks are dysregulated during tumourigenesis. The trimethylation of histone H3 at lysine 36 (H3K36me3) and SETD2, the enzyme solely responsible for this histone mark, are important in many cellular processes and have been reported to be lost or mutated in cancers. Targeting cells that are deficient in H3K36me3 or SETD2 is therefore an attractive therapeutic strategy for specifically killing tumour cells with minimal effect on normal tissue using the concept of synthetic lethality. Using a previously reported synthetic lethal interaction between SETD2 and the WEE1 kinase as a starting point, a high-throughput small-molecule compound screen was performed in cells in which SETD2 had been deleted using CRISPR-Cas9 technology. The aim of this screen was to identify two categories of hits: (1) those that enhance the SETD2-WEE1 synthetic lethality (synergy hits) and (2) those that target SETD2-CRISPR cells on their own (novel synthetic lethality hits). Analysis of the synergy hits focused on histone deacetylases (HDACs), which revealed that the Class I HDACs HDAC1 and HDAC2 had differential effects on the SETD2-WEE1 synthetic lethality. Furthermore, depletion of both HDACs individually led to a significant loss of viability specifically in SETD2-CRISPR cells. The results suggest a putative synthetic lethality between SETD2 and HDAC2 that bears certain similarities to the relationship between SETD2 and WEE1. The most potent among the novel synthetic lethality hits was a compound called RITA, which demonstrated the ability to target SETD2 deficiency in the form of CRISPR knockout, siRNA knockdown, and naturally occurring mutations. This was accompanied by a striking upregulation and activation of the p53 tumour suppressor, activation of the DNA damage response, cell cycle arrest, and apoptosis. These phenotypes were further associated with increased levels of replication stress markers and reduced replication fork velocity. The potential mechanism of action of this compound was investigated via a number of strategies based on current knowledge of SETD2 and RITA, as well as more exploratory approaches. Overall, the data indicate that perturbations in the G1/S transition and replication stress play key roles in mediating RITA’s cytotoxicity in the context of SETD2 loss.</p
Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In ... more Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFβ), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFβ is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFβ signaling. HPV blockade of TGFβ signaling, or pharmaceutical TGFβ inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFβ inhibitors for optimal therapeutic combinations that improve patient outcome.
The present study used ERPs to provide precise temporal information about the modulation of maske... more The present study used ERPs to provide precise temporal information about the modulation of masked repetition priming effects by word frequency during the course of target word recognition. Contrary to the pattern seen with behavioral response times in prior research, we predicted that high-frequency words should generate larger and earlier peaking repetition priming effects than low-frequency words in the N400 time window. This prediction was supported by the results of two experiments. Furthermore, repetition priming effects in the N250 time window were found for low-frequency words in both experiments, whereas for high-frequency words these effects were only seen at the shorter (50 ms) SOA used in Experiment 2, and not in Experiment 1 (70 ms SOA). We explain this pattern as resulting from reset mechanisms operating on the form representations activated by prime stimuli when primes and targets are processed as separate perceptual events.
Stem cells and cancer stem cells show a distinct proliferative potential for forming clonal float... more Stem cells and cancer stem cells show a distinct proliferative potential for forming clonal floating colonies in vitro, termed tumor spheres. We have generated tumor spheres from a clinical gastric cancer sample (GCIC38) using serum-free culture, and expression of EpCAM and cytokeratin proteins confirmed the epithelial origin of these cells. GCIC38 sphere-derived cells were able to initiate tumors in NOD/SCID mice that recapitulate the tumor in the gastric cancer patient. Real-time PCR analysis showed that in contrast to human gastric cancer cell lines AGS, SNU16 and NCI-N87, GCIC38 sphere-derived cells express relatively high levels of stem cell markers such as SOX2, Musashi-1, and Bmi-1. Surprisingly, mRNA expression of the well-known tumor suppressor for gastric cancer, RUNX3, was found to be 4.6 and 16-fold higher than that of SNU16 and NCI-N87 cells respectively. Consistently, western blot analyses showed that GCIC38 sphere-derived cells express high levels of RUNX3 compared to a panel of gastric cancer cell lines. Immunofluorescence staining confirmed that RUNX3 was expressed in the nuclei of these cells, ruling out the possibility of RUNX3 protein mislocalization. To further characterize RUNX3 in GCIC38 sphere-derived gastric cancer cells, we cloned and identified a novel truncated 0.4kb RUNX3 variant (RUNX3-LG) that is expressed concurrently with the 1.2kb full length RUNX3 transcript (wt-RUNX3) in these cells. Sequence analysis revealed that RUNX3-LG is a truncated form of RUNX3 variant 2 encoded by a transcript utilizing the P2 promoter. There are no mutations in the wt-RUNX3 transcript whilst exons 3, 4 and 5 are spliced out in the RUNX3-LG transcript resulting in the deletion of RUNT domain in the RUNX3-LG protein. Immunoprecipitation studies using RUNX3 antibodies showed endogenous expression of a 15kDa protein in GCIC38 sphere-derived cells, concordant with the predicted size of RUNX3-LG protein. Preliminary data show that ectopic over-expression of RUNX3-LG in AGS gastric cancer cells results in reduced expression of RUNX3 target genes Claudin-1, S100A2 and p21. We propose that RUNX3-LG may be a dominant negative variant in gastric cancer cells with high wt-RUNX3 expression, which may abrogate wt-RUNX3 gene function including its tumor-suppressive effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2011-4033
PDF file - 126KB, Supplementary Figure S2 shows characterization of CD44v8-10 antibody by flow cy... more PDF file - 126KB, Supplementary Figure S2 shows characterization of CD44v8-10 antibody by flow cytometry and gating strategy and isotype control for Figure 3.
The surface marker CD44 has been identified as one of several markers associated with cancer stem... more The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers. Cancer Res; 74(9); 2630-41. Ó2014 AACR.
PDF file - 118KB, Supplementary Figure S1 shows gating strategies for flow cytometry, quantitatio... more PDF file - 118KB, Supplementary Figure S1 shows gating strategies for flow cytometry, quantitation for EpCAM expression level and EpCAM+ cells initiated tumors in mice.
PDF file - 20KB, Supplementary Table S4 shows p53 mutation status and proportion of CD44+ cells i... more PDF file - 20KB, Supplementary Table S4 shows p53 mutation status and proportion of CD44+ cells in gastric tumor xenografts.
PDF file - 24KB, Supplementary Table S3 shows effects of CD44 variant over-expression and knockdo... more PDF file - 24KB, Supplementary Table S3 shows effects of CD44 variant over-expression and knockdown on tumor initiation potential.
PDF file - 93KB, Supplementary Figure S4 shows gating strategy for sorting of CD44 depleted TMK1 ... more PDF file - 93KB, Supplementary Figure S4 shows gating strategy for sorting of CD44 depleted TMK1 cells for tumor initiation assay and xenograft tumors formed.
PDF file - 130KB, Supplementary Figure S3 shows fractionation of CD44v+ cells for limiting diluti... more PDF file - 130KB, Supplementary Figure S3 shows fractionation of CD44v+ cells for limiting dilution analysis in vitro and in vivo.
PDF file - 25KB, Supplementary Table S2 shows estimation of CSC frequency in unsorted, EpCAM+CD13... more PDF file - 25KB, Supplementary Table S2 shows estimation of CSC frequency in unsorted, EpCAM+CD133+ and EpCAM+CD133- fractions from GC16 xenografts.
The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (... more The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in certain cancer types, identifying SETD2 as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to the compound RITA, resulting in significant p53 induction and apoptosis. This is further associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. RITA sensitivity is linked to the phenol sulphotransferase SULT1A1, which we find to be highly upregulated in cells that lack SETD2. Depletion of SULT1A1 or addition of the phenol sulphotransferase inhibitor DCNP abolishes these phenotypes and suppresses the sensitivity of SETD2-deficient cancer cells, identifying SULT1A1 activity to be critical in mediating the potent cytotoxicity of RITA against SETD2-deficient cells. These findings define ...
The surfacemarker CD44 has been identified as one of several markers associated with cancer stem ... more The surfacemarker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues.We identified CD44v8-10 as the predominantCD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Recipro...
Histone modifications play diverse and essential roles in the maintenance of genome integrity, as... more Histone modifications play diverse and essential roles in the maintenance of genome integrity, as evidenced by the frequency at which different histone marks are dysregulated during tumourigenesis. The trimethylation of histone H3 at lysine 36 (H3K36me3) and SETD2, the enzyme solely responsible for this histone mark, are important in many cellular processes and have been reported to be lost or mutated in cancers. Targeting cells that are deficient in H3K36me3 or SETD2 is therefore an attractive therapeutic strategy for specifically killing tumour cells with minimal effect on normal tissue using the concept of synthetic lethality. Using a previously reported synthetic lethal interaction between SETD2 and the WEE1 kinase as a starting point, a high-throughput small-molecule compound screen was performed in cells in which SETD2 had been deleted using CRISPR-Cas9 technology. The aim of this screen was to identify two categories of hits: (1) those that enhance the SETD2-WEE1 synthetic lethality (synergy hits) and (2) those that target SETD2-CRISPR cells on their own (novel synthetic lethality hits). Analysis of the synergy hits focused on histone deacetylases (HDACs), which revealed that the Class I HDACs HDAC1 and HDAC2 had differential effects on the SETD2-WEE1 synthetic lethality. Furthermore, depletion of both HDACs individually led to a significant loss of viability specifically in SETD2-CRISPR cells. The results suggest a putative synthetic lethality between SETD2 and HDAC2 that bears certain similarities to the relationship between SETD2 and WEE1. The most potent among the novel synthetic lethality hits was a compound called RITA, which demonstrated the ability to target SETD2 deficiency in the form of CRISPR knockout, siRNA knockdown, and naturally occurring mutations. This was accompanied by a striking upregulation and activation of the p53 tumour suppressor, activation of the DNA damage response, cell cycle arrest, and apoptosis. These phenotypes were further associated with increased levels of replication stress markers and reduced replication fork velocity. The potential mechanism of action of this compound was investigated via a number of strategies based on current knowledge of SETD2 and RITA, as well as more exploratory approaches. Overall, the data indicate that perturbations in the G1/S transition and replication stress play key roles in mediating RITA’s cytotoxicity in the context of SETD2 loss.</p
Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In ... more Repair of DNA damage protects genomic integrity, which is key to tissue functional integrity. In cancer, the type and fidelity of DNA damage response is the fundamental basis for clinical response to cytotoxic therapy. Here we consider the contribution of transforming growth factor-beta (TGFβ), a ubiquitous, pleotropic cytokine that is abundant in the tumor microenvironment, to therapeutic response. The action of TGFβ is best illustrated in head and neck squamous cell carcinoma (HNSCC). Survival of HNSCC patients with human papilloma virus (HPV) positive cancer is more than double compared to those with HPV-negative HNSCC. Notably, HPV infection profoundly impairs TGFβ signaling. HPV blockade of TGFβ signaling, or pharmaceutical TGFβ inhibition that phenocopies HPV infection, shifts cancer cells from error-free homologous-recombination DNA double-strand-break (DSB) repair to error-prone alternative end-joining (altEJ). Cells using altEJ are more sensitive to standard of care radiotherapy and cisplatin, and are sensitized to PARP inhibitors. Hence, HPV-positive HNSCC is an experiment of nature that provides a strong rationale for the use of TGFβ inhibitors for optimal therapeutic combinations that improve patient outcome.
The present study used ERPs to provide precise temporal information about the modulation of maske... more The present study used ERPs to provide precise temporal information about the modulation of masked repetition priming effects by word frequency during the course of target word recognition. Contrary to the pattern seen with behavioral response times in prior research, we predicted that high-frequency words should generate larger and earlier peaking repetition priming effects than low-frequency words in the N400 time window. This prediction was supported by the results of two experiments. Furthermore, repetition priming effects in the N250 time window were found for low-frequency words in both experiments, whereas for high-frequency words these effects were only seen at the shorter (50 ms) SOA used in Experiment 2, and not in Experiment 1 (70 ms SOA). We explain this pattern as resulting from reset mechanisms operating on the form representations activated by prime stimuli when primes and targets are processed as separate perceptual events.
Stem cells and cancer stem cells show a distinct proliferative potential for forming clonal float... more Stem cells and cancer stem cells show a distinct proliferative potential for forming clonal floating colonies in vitro, termed tumor spheres. We have generated tumor spheres from a clinical gastric cancer sample (GCIC38) using serum-free culture, and expression of EpCAM and cytokeratin proteins confirmed the epithelial origin of these cells. GCIC38 sphere-derived cells were able to initiate tumors in NOD/SCID mice that recapitulate the tumor in the gastric cancer patient. Real-time PCR analysis showed that in contrast to human gastric cancer cell lines AGS, SNU16 and NCI-N87, GCIC38 sphere-derived cells express relatively high levels of stem cell markers such as SOX2, Musashi-1, and Bmi-1. Surprisingly, mRNA expression of the well-known tumor suppressor for gastric cancer, RUNX3, was found to be 4.6 and 16-fold higher than that of SNU16 and NCI-N87 cells respectively. Consistently, western blot analyses showed that GCIC38 sphere-derived cells express high levels of RUNX3 compared to a panel of gastric cancer cell lines. Immunofluorescence staining confirmed that RUNX3 was expressed in the nuclei of these cells, ruling out the possibility of RUNX3 protein mislocalization. To further characterize RUNX3 in GCIC38 sphere-derived gastric cancer cells, we cloned and identified a novel truncated 0.4kb RUNX3 variant (RUNX3-LG) that is expressed concurrently with the 1.2kb full length RUNX3 transcript (wt-RUNX3) in these cells. Sequence analysis revealed that RUNX3-LG is a truncated form of RUNX3 variant 2 encoded by a transcript utilizing the P2 promoter. There are no mutations in the wt-RUNX3 transcript whilst exons 3, 4 and 5 are spliced out in the RUNX3-LG transcript resulting in the deletion of RUNT domain in the RUNX3-LG protein. Immunoprecipitation studies using RUNX3 antibodies showed endogenous expression of a 15kDa protein in GCIC38 sphere-derived cells, concordant with the predicted size of RUNX3-LG protein. Preliminary data show that ectopic over-expression of RUNX3-LG in AGS gastric cancer cells results in reduced expression of RUNX3 target genes Claudin-1, S100A2 and p21. We propose that RUNX3-LG may be a dominant negative variant in gastric cancer cells with high wt-RUNX3 expression, which may abrogate wt-RUNX3 gene function including its tumor-suppressive effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2011-4033
PDF file - 126KB, Supplementary Figure S2 shows characterization of CD44v8-10 antibody by flow cy... more PDF file - 126KB, Supplementary Figure S2 shows characterization of CD44v8-10 antibody by flow cytometry and gating strategy and isotype control for Figure 3.
The surface marker CD44 has been identified as one of several markers associated with cancer stem... more The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers. Cancer Res; 74(9); 2630-41. Ó2014 AACR.
Uploads
Papers by Kirsten Lopez