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Colorectal cancer continues to be a major health issue even though screening methods have facilitated early detection. Despite the high sensitivity of white-light colonoscopy, it frequently overlooks invasive flat or depressed lesions, which can lead to the development of larger, advanced tumors. Fluorescence molecular imaging offers a promising approach for early tumor detection by targeting specific molecular characteristics of lesions. CD24 is upregulated during the adenoma-to-colorectal cancer transition, providing a potential target for fluorescence molecular imaging. In this study, we developed a second near-infrared window (NIR-II) fluorescent probe with a high affinity for CD24 and evaluated its efficacy and targeting ability in cellular models, murine models, and clinical samples of colorectal cancer. CD24 expression was elevated in 76% of adenomas and 80% of colorectal cancers. In a colitis-associated cancer mouse model, NIR-II imaging with the CD24-targeted probe achieved a significantly higher tumor-to-background ratio compared with conventional NIR-I imaging. The probe demonstrated exceptional sensitivity (92%) and specificity (92%) for detecting colorectal cancer, including small lesions less than 1 mm in size. This led to the identification of precancerous lesions missed by white-light detection and lesions missed by NIR-I imaging. Moreover, ex vivo human tissue incubation with the probe supported the potential for intraprocedural lesion identification via topical probe application during colonoscopy. In conclusion, this study successfully demonstrates the potential of CD24-targeted NIR-II imaging for identifying colorectal neoplasia, highlighting its significance for early colorectal cancer detection in the gastrointestinal tract. Significance: Overexpression of CD24 in colorectal dysplasia provides the opportunity to use an NIR-II fluorescent probe targeting CD24 to detect colorectal neoplasms, including invisible lesions that are missed by white-light colonoscopy., (©2024 American Association for Cancer Research.)

Background: According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in noncancerous breast tissue., Methods: The analysis included 414 women with biopsy-confirmed benign breast disease in the Nurses' Health Study and Nurses' Health Study II. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaires. IHC staining of stem cell markers (CD44, CD24, and aldehyde dehydrogenase family 1 member A1) in histopathologically normal epithelial and stromal breast tissue was quantified using an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders., Results: Birthweight [≥10.0 vs. <5.5 lbs: β (95% confidence interval) = 4.29 (1.02, 7.56); P trend = 0.001 in the stroma] and adult height [≥67.0 vs. <63.0 inch: 0.86 (0.14, 1.58); P trend = 0.02 in the epithelium and stroma combined] were positively associated with CD44 expression. Childhood body fatness was inversely associated (P trend = 0.03) whereas adult height was positively associated with CD24 expression in combined stroma and epithelium (P trend = 0.03)., Conclusions: Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with benign breast disease., Impact: Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast., (©2024 American Association for Cancer Research.)

NAD-dependent deacetylase sirtuin-1 (SIRT1) is a class III histone deacetylase that positively regulates cancer-related pathways such as proliferation and stress resistance. SIRT1 has been shown to promote progression of colorectal cancer and is associated with cancer stemness, yet the precise mechanism between colorectal cancer stemness and SIRT1 remains to be further clarified. Here we report that SIRT1 signaling regulates colorectal cancer stemness by enhancing expression of CD24, a colorectal cancer stemness promoter. A novel miRNA, miR-1185-1, suppressed the expression of CD24 by targeting its 3'UTR (untranslated region) and could be inhibited by SIRT1 via histone deacetylation. Targeting SIRT1 by RNAi led to elevated H3 lysine 9 acetylation on the promoter region of miR-1185-1, which increased expression of miR-1185-1 and further repressed CD24 translation and colorectal cancer stemness. In a mouse xenograft model, overexpression of miR-1185-1 in colorectal cancer cells substantially reduced tumor growth. In addition, expression of miR-1185-1 was downregulated in human colorectal cancer tissues, whereas expression of CD24 was increased. In conclusion, this study not only demonstrates the essential roles of a SIRT1-miR-1185-1-CD24 axis in both colorectal cancer stemness properties and tumorigenesis but provides a potential therapeutic target for colorectal cancer treatment. SIGNIFICANCE: A novel tumor suppressor miR-1185-1 is involved in molecular regulation of CD24- and SIRT1-related cancer stemness networks, marking it a potential therapeutic target in colorectal cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/23/5257/F1.large.jpg., (©2020 American Association for Cancer Research.)

The surface glycoprotein THY is a marker of myoepithelial precursor cells, which are basal cells with epithelial-mesenchymal intermediate phenotype originating from the ectoderm. Myoepithelial precursor cells are lost during progression from in situ to invasive carcinoma. To define the functional role of Thy1-positive cells within the myoepithelial population, we tracked Thy1 expression in human breast cancer samples, isolated THY1-positive myoepithelial progenitor cells (CD44 + /CD24 low /CD90 + ), and established long-term cultures (parental cells). Parental cells were used to generate a xenograft model to examine Thy1 expression during tumor formation. Post-transplantation cell cultures lost THY 1 expression through methylation at the THY1 locus and this is associated with an increase in EGFR and NOTCH1 transcript levels. Thy1-low cells are sensitive to the EGFR/HER2 dual inhibitor lapatinib. High THY1 expression is associated with poorer relapse-free survival in patients with breast cancer. THY1 methylation may track the shift of bipotent progenitors into differentiated cells. Thy1 is a good candidate biomarker in basal-like breast cancer. IMPLICATIONS: Our findings provide evidence that THY1 expression is lost in xenografts due to promoter methylation. Thy1-low cells with increased EGFR and Notch1 expression are responsive to target therapy. Because DNA methylation is often altered in early cancer development, candidate methylation markers may be exploited as biomarkers for basal-like breast cancer., (©2018 American Association for Cancer Research.)

Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a coculture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAM) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this de novo P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling underflow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion ex vivo and ID8 adhesion in vivo Analysis of samples from patients with HGSOC confirmed increased MIP-1β and P-selectin, suggesting that this novel multicellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti-CCR5 and P-selectin therapies developed for other indications. Significance: This study reports novel insights on the peritoneal dissemination occurring during progression of ovarian cancer and has potential for therapeutic intervention. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg Cancer Res; 78(13); 3560-73. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Elevated tumor expression of the cell surface GPI-linked CD24 protein signals poor patient prognosis in many tumor types. However, some cancer cells selected to be negative for surface CD24 (surCD24 - ) still retain aggressive phenotypes in vitro and in vivo Here, we resolve this apparent paradox with the discovery of biologically active, nuclear CD24 (nucCD24) and finding that its levels are unchanged in surCD24 - cells. Using the complementary techniques of biochemical cellular fractionation and immunofluorescence, we demonstrate a signal for CD24 in the nucleus in cells from various histologic types of cancer. Nuclear-specific expression of CD24 (NLS-CD24) increased anchorage-independent growth in vitro and tumor formation in vivo Immunohistochemistry of patient tumor samples revealed the presence of nucCD24, whose signal intensity correlated positively with the presence of metastatic disease. Analysis of gene expression between cells expressing CD24 and NLS-CD24 revealed a unique nucCD24 transcriptional signature. The median score derived from this signature was able to stratify overall survival in four patient datasets from bladder cancer and five patient datasets from colorectal cancer. Patients with high scores (more nucCD24-like) had reduced survival. These findings define a novel and functionally important intracellular location of CD24; they explain why surCD24 - cells can remain aggressive, and they highlight the need to consider nucCD24 in both fundamental research and therapeutic development. Cancer Res; 77(18); 4858-67. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Cancer tissue functions as an ecosystem of a diverse set of cells that interact in a complex tumor microenvironment. Genomic tools applied to biopsies in bulk fail to account for this tumor heterogeneity, whereas single-cell imaging methods limit the number of cells which can be assessed or are very resource intensive. The current study presents methods based on flow cytometric analysis and cell sorting using known cell surface markers (CXCR4/CD184, CD24, THY1/CD90) to identify and interrogate distinct groups of cells in triple-negative breast cancer clinical biopsy specimens from patient-derived xenograft (PDX) models. The results demonstrate that flow cytometric analysis allows a relevant subgrouping of cancer tissue and that sorting of these subgroups provides insights into cancer cell populations with unique, reproducible, and functionally divergent gene expression profiles. The discovery of a drug resistance signature implies that uncovering the functional interaction between these populations will lead to deeper understanding of cancer progression and drug response. Implications: PDX-derived human breast cancer tissue was investigated at the single-cell level, and cell subpopulations defined by surface markers were identified which suggest specific roles for distinct cellular compartments within a solid tumor. Mol Cancer Res; 15(4); 429-38. ©2016 AACR ., (©2016 American Association for Cancer Research.)

In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175-85. ©2016 AACR., Competing Interests: The authors declared no conflict of interest., (©2016 American Association for Cancer Research.)

Survival rates associated with pancreatic cancer remain dismal despite advancements in detection and experimental treatment strategies. Genetically engineered mouse models of pancreatic tumorigenesis have gained considerable attention based on their ability to recapitulate key clinical features of human disease including chemotherapeutic resistance and fibrosis. However, it is unclear if transgenic systems exemplified by the Kras(G12D)/Trp53(R172H)/Pdx-1-Cre (KPC) mouse model recapitulate the functional heterogeneity of human pancreatic tumors harboring distinct cells with tumorigenic properties. To facilitate tracking of heterogeneous tumor cell populations, we incorporated a luciferase-based tag into the genetic background of the KPC mouse model. We isolated pancreatic cancer cells from multiple independent tumor lines and found that roughly 1 out of 87 cells exhibited tumorigenic capability. Notably, this frequency is significantly higher than reported for human pancreatic adenocarcinomas. Cancer stem cell (CSC) markers, including CD133, CD24, Sca-1, and functional Aldefluor activity, were unable to discriminate tumorigenic from nontumorigenic cells in syngeneic transplants. Furthermore, three-dimensional spheroid cultures originating from KPC tumors did not enrich for cells with stem-like characteristics and were not significantly more tumorigenic than cells cultured as monolayers. Additionally, we did not observe significant differences in response to gemcitabine or salinomycin in several isolated subpopulations. Taken together, these studies show that the hierarchical organization of CSCs in human disease is not recapitulated in a commonly used mouse model of pancreatic cancer and therefore provide a new view of the phenotypic and functional heterogeneity of tumor cells., (©2015 American Association for Cancer Research.)

Unlabelled: Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44(+)/CD24(low/-) population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer., Implications: Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy., (©2015 American Association for Cancer Research.)

Transcriptional control is the major determinant of cell fate. The steroid receptor coactivator (SRC)-1 enhances the activity of the estrogen receptor in breast cancer cells, where it confers cell survival benefits. Here, we report that a global analysis of SRC-1 target genes suggested that SRC-1 also mediates transcriptional repression in breast cancer cells. Combined SRC-1 and HOXC11 ChIPseq analysis identified the differentiation marker, CD24, and the apoptotic protein, PAWR, as direct SRC-1/HOXC11 suppression targets. Reduced expression of both CD24 and PAWR was associated with disease progression in patients with breast cancer, and their expression was suppressed in metastatic tissues. Investigations in endocrine-resistant breast cancer cell lines and SRC-1(-/-)/PyMT mice confirmed a role for SRC-1 and HOXC11 in downregulation of CD24 and PAWR. Through bioinformatic analysis and liquid chromatography/mass spectrometry, we identified AP1 proteins and Jumonji domain containing 2C (JMD2C/KDM4C), respectively, as members of the SRC-1 interactome responsible for transcriptional repression. Our findings deepen the understanding of how SRC-1 controls transcription in breast cancers., (©2014 AACR.)

A major barrier to vaccines in cancer treatment is their failure to activate and maintain a complete cancer-specific CD8(+) effector T-cell repertoire. Low-avidity T cells are more likely to escape clonal deletion in the thymus when compared with high-avidity T cells, and therefore comprise the major population of effector T cells available for activation in patients with cancer. However, low-avidity T cells fail to traffic into the tumor microenvironment and function in eradicating tumor under optimal vaccination conditions as opposed to high-avidity T cells that escape clonal deletion and function in tumor killing. We used high- and low-avidity T-cell receptor transgenic CD8(+) T cells specific for the immunodominant epitope HER2/neu (RNEU420-429) to identify signaling pathways responsible for the inferior activity of the low-avidity T cells. Adoptive transfer of these cells into tumor-bearing vaccinated mice identified the members of apoptosis pathways that are upregulated in low-avidity T cells. The increased expression of proapoptotic proteins by low-avidity T cells promoted their own cell death and also that of other tumor-specific CD8(+) T cells within their local environment. Importantly, we show that this proapoptotic effect can be overcome by using a strong costimulatory signal that prevents the activation-induced cell death and enables the low-avidity T cells to traffic into the tumor and assist in tumor clearance. These findings identify new therapeutic opportunities for activating the most potent anticancer T-cell responses.

Accumulating evidence suggests that Notch3 (N3) is involved in breast cancer development, but its precise contributions are not well understood. Here, we report that pregnant mice expressing an activated intracellular form of N3 (N3(IC)) exhibit a cyclin D1-dependent expansion of premalignant CD24(+) CD29(low) luminal progenitors with enhanced differentiation potential in vitro and in vivo. Parous mice developed luminal mammary tumors in a cyclin D1-dependent manner. Notably, mice expressing higher levels of N3(IC) exhibited tumors resembling inflammatory breast cancer that frequently metastasized. N3(IC)-induced tumors contained a large percentage of tumor-initiating cells, but these were reduced significantly in tumors derived from N3(IC) transgenic mice that were heterozygous for cyclin D1. After transplantation in the presence of normal mammary cells, N3(IC)-expressing tumor cells became less malignant, differentiating into CK6(+) CK18(+) CK5(-) alveolar-like structures akin to expanded luminal progenitors from which they were likely derived. Taken together, our results argue that activated N3 signaling primarily affects luminal progenitors among mammary cell subsets, with more pronounced levels of activation influencing tumor type, and provide a novel model of inflammatory breast cancer.

Expression of cyclin E proteolytic cleavage products, low-molecular weight cyclin E (LMW-E), is associated with poor clinical outcome in patients with breast cancer and it enhances tumorigenecity in mouse models. Here we report that LMW-E expression in human mammary epithelial cells induces an epithelial-to-mesenchymal transition phenotype, increases the CD44(hi)/CD24(lo) population, enhances mammosphere formation, and upregulates aldehyde dehydrogenase expression and activity. We also report that breast tumors expressing LMW-E have a higher proportion of CD44(hi)/CD24(lo) tumor cells as compared with tumors expressing only full-length cyclin E. In order to explore how LMW-E enriches cancer stem cells in breast tumors, we conducted a protein microarray analysis that identified the histone acetyltransferase (HAT) Hbo1 as a novel cyclin E/CDK2 substrate. The LMW-E/CDK2 complex phosphorylated Hbo1 at T88 without affecting its HAT activity. When coexpressed with LMW-E/CDK2, wild-type Hbo1 promoted enrichment of cancer stem-like cells (CSC), whereas the T88 Hbo1 mutant reversed the CSC phenotype. Finally, doxorubicin and salinomycin (a CSC-selective cytotoxic agent) synergized to kill cells expressing LMW-E, but not full-length cyclin E. Collectively, our results suggest that the heightened oncogenecity of LMW-E relates to its ability to promote CSC properties, supporting the design of therapeutic strategies to target this unique function., (©2013 AACR.)

Unlabelled: Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer (LBC) cells and patient tumor specimens (LBC "instigators") establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors ("responders"). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGF receptor 2(+) tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically., Significance: Currently, processes that mediate progression of otherwise indolent tumors are not well understood, making it difficult to accurately predict which cancer patients are likely to relapse. Our findings highlight the macroenvironment as an important component of disease progression that can be exploited to more accurately identify patients who would benefit from adjuvant therapy., (©2012 AACR.)

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity., (©2011 AACR.)

Metastasis is lethal in most bladder cancer patients. Expression of CD24, a glycosyl phosphatidylinositol (GPI)-linked sialoglycoprotein and cancer stem cell marker, is associated with metastatic progression in multiple cancer types, yet the role of CD24 in this process remains unclear. While developing a murine model of human metastatic bladder cancer, we observed that tumor cell CD24 expression correlated with a propensity to metastasize to the lung. Our immunohistochemical evaluation of 60 paired primary and metastatic human bladder cancer samples revealed increased intensity (P < 0.001) and frequency (P < 0.001) of CD24 expression in metastases. To directly evaluate the role of CD24 in metastatic colonization, we manipulated CD24 expression in human bladder cancer cell lines using short hairpin RNA depletion, cDNA overexpression, and fluorescence-activated cell sorting selection. Although suppression of CD24 reduced acute tumor cell retention in the lungs of mice inoculated intravenously with cancer cells, this differential retention was no longer apparent after 24 hours, prompting us to evaluate the role of CD24 in lung colonization. Here, CD24 was found necessary for subsequent development of lung metastases. We next treated clinically detectable lung metastases in mice with anti-CD24 antibody and observed reduced tumor growth and prolonged survival. These findings suggest that CD24 is a lynchpin of metastatic progression and a promising therapeutic target for antimetastatic therapy.

We report a novel function for the CD24 molecule in pancreatic cancer cells. Intracellular CD24 is associated with stress granules that contain specific mRNAs and RNA-binding proteins that regulate mRNA stability and translation. Intracellular CD24 in stress granules is associated with G3BP, a phosphorylation-dependent endoribonuclease. The vesicles in which the CD24/G3BP complex localizes are transported toward cell protrusions in migrating cells. We show that G3BP binds to and degrades Binder of Arl Two (BART) mRNA. BART was originally identified as a binding partner of ARL2, a small G-protein implicated as a regulator of microtubule dynamics and folding. Intracellular CD24 inhibits the specific endoribonuclease activity of G3BP toward BART mRNA in stress granules. We show that knockdown of CD24 increases retroperitoneal invasion and liver metastasis of pancreatic cancer cells in an orthotopic xenograft model, and that BART also prevents retroperitoneal invasion and liver metastasis of pancreatic cancer cells. Our results imply that surface CD24 may play a role in the inhibition of cell invasion and metastasis, and that intracellular CD24 inhibits invasiveness and metastasis through its influence on the posttranscriptional regulation of BART mRNA levels via G3BP RNase activity.

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44(high)CD24(low) "cancer stem cell-like" phenotype and expressed significantly less HER2 compared with non-stem cells. Likewise, the CD44(high)CD24(low) population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44(high)CD24(low) cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants.

The family of GLI zinc finger transcription factors regulates the expression of genes involved in many important cellular processes, notably embryonal development and cellular differentiation. The glioma-associated oncogene homologue 1 (GLI1) isoform, in particular, has attracted much attention because of its frequent activation in many human cancers and its interactions with other signaling pathways, such as those mediated by K-RAS, transforming growth factor-beta, epidermal growth factor receptor, and protein kinase A. Here, we report the identification of a novel truncated GLI1 splice variant, tGLI1, with an in-frame deletion of 123 bases (41 codons) spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Expression of tGLI1 is undetectable in normal cells but is high in glioblastoma multiforme (GBM) and other cancer cells. Although tGLI1 undergoes nuclear translocalization and transactivates GLI1-binding sites similar to GLI1, unlike GLI1, it is associated with increased motility and invasiveness of GBM cells. Using microarray analysis, we showed >100 genes to be differentially expressed in tGLI1-expressing compared with GLI1-expressing GBM cells, although both cell types expressed equal levels of known GLI1-regulated genes, such as PTCH1. We further showed one of the tGLI1 up-regulated genes, CD24, an invasion-associated gene, to be required for the migratory and invasive phenotype of GBM cells. These data provide conclusive evidence for a novel gain-of-function GLI1 splice variant that promotes migration and invasiveness of GBM cells and open up a new research paradigm on the role of the GLI1 pathway in malignancy.

Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a Lin(-)CD29(H)CD24(H) subpopulation of tumor-initiating cells. Upon subsequent transplantation, this subpopulation generated heterogeneous tumors that displayed properties similar to the primary tumor. Analysis of biomarkers suggests the Lin(-)CD29(H)CD24(H) subpopulation may have arisen from a bipotent mammary progenitor. Differentially expressed genes in the Lin(-)CD29(H)CD24(H) mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. These studies provide in vitro and in vivo data that support the cancer stem cell (CSC) hypothesis. Furthermore, this p53-null mouse mammary tumor model may allow us to identify new CSC markers and to test the functional importance of these markers.

Ral GTPases are important mediators of transformation, tumorigenesis, and cancer progression. We recently identified the metastasis-associated protein CD24, a glycosyl phosphatidyl inositol-linked surface protein, as a downstream target of Ral signaling by profiling the expression of RalA/B-depleted bladder carcinoma cells. Because CD24 is highly expressed in bladder and many other tumor types, we sought to determine if this protein plays an essential role in maintaining the malignant phenotype. Here, we show that loss of CD24 function in cell lines derived from common tumor types is associated with decreased rates of cell proliferation, clonogenicity in soft agar, changes in the actin cytoskeleton, and induction of apoptosis. Given these phenotypes, we evaluated a human bladder cancer tissue microarray by immunohistochemistry for CD24 to determine if CD24 is a prognostic cancer biomarker. Multivariate analysis showed that increased CD24 expression correlated with shorter patient disease-free survival (P = 0.07). In conclusion, we show that CD24 is a novel and functionally relevant Ral-regulated target and a potentially important prognostic marker. We suggest that these insights may lead to future therapeutic approaches that seek to eliminate CD24 function in cancer cells.