Loss of PDPK1 abrogates resistance to gemcitabine in label-retaining pancreatic cancer cells.

BACKGROUND: Label-retaining cancer cells (LRCC) have been proposed as a model of slowly cycling cancer stem cells (CSC) which mediate resistance to chemotherapy, tumor recurrence, and metastasis. The molecular mechanisms of chemoresistance in LRCC remain to-date incompletely understood. This study aims to identify molecular targets in LRCC that can be exploited to overcome resistance to gemcitabine, a standard chemotherapy agent for the treatment of pancreas cancer. METHODS: LRCC were isolated following Cy5-dUTP staining by flow cytometry from pancreatic cancer cell lines. Gene expression profiles obtained from LRCC, non-LRCC (NLRCC), and bulk tumor cells were used to generate differentially regulated pathway networks. Loss of upregulated targets in LRCC on gemcitabine sensitivity was assessed via RNAi experiments and pharmacological inhibition. Expression patterns of PDPK1, one of the upregulated targets in LRCC, was studied in patients' tumor samples and correlated with pathological variables and clinical outcome. RESULTS: LRCC are significantly more resistant to gemcitabine than the bulk tumor cell population. Non-canonical EGF (epidermal growth factor)-mediated signal transduction emerged as the top upregulated network in LRCC compared to non-LRCC, and knock down of EGF signaling effectors PDPK1 (3-phosphoinositide dependent protein kinase-1), BMX (BMX non-receptor tyrosine kinase), and NTRK2 (neurotrophic receptor tyrosine kinase 2) or treatment with PDPK1 inhibitors increased growth inhibition and induction of apoptosis in response to gemcitabine. Knockdown of PDPK1 preferentially increased growth inhibition and reduced resistance to induction of apoptosis upon gemcitabine treatment in the LRCC vs non-LRCC population. These findings are accompanied by lower expression levels of PDPK1 in tumors compared to matched uninvolved pancreas in surgical resection specimens and a negative association of membranous localization on IHC with high nuclear grade (p < 0.01). CONCLUSION: Pancreatic cancer cell-derived LRCC are relatively resistant to gemcitabine and harbor a unique transcriptomic profile compared to bulk tumor cells. PDPK1, one of the members of an upregulated EGF-signaling network in LRCC, mediates resistance to gemcitabine, is found to be dysregulated in pancreas cancer specimens, and might be an attractive molecular target for combination therapy studies.

Label-retaining liver cancer cells are relatively resistant to sorafenib.

OBJECTIVE: The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label-retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). The objective was to test whether LRCC are resistant to sorafenib. METHODS: We tested human HCC derived LRCC and non-LRCC before and after treatment with sorafenib. RESULTS: LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non-LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless-type MMTV-integration-site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non-uniform activation of specific isoforms of the sorafenib target proteins extracellular-signal-regulated kinases and v-akt-murine-thymoma-viral-oncogene homologue (AKT) in LRCC but not in non-LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. CONCLUSIONS: Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.

Tumor-initiating label-retaining cancer cells in human gastrointestinal cancers undergo asymmetric cell division.

Label-retaining cells (LRCs) have been proposed to represent adult tissue stem cells. LRCs are hypothesized to result from either slow cycling or asymmetric cell division (ACD). However, the stem cell nature and whether LRC undergo ACD remain controversial. Here, we demonstrate label-retaining cancer cells (LRCCs) in several gastrointestinal (GI) cancers including fresh surgical specimens. Using a novel method for isolation of live LRCC, we demonstrate that a subpopulation of LRCC is actively dividing and exhibits stem cells and pluripotency gene expression profiles. Using real-time confocal microscopic cinematography, we show live LRCC undergoing asymmetric nonrandom chromosomal cosegregation LRC division. Importantly, LRCCs have greater tumor-initiating capacity than non-LRCCs. Based on our data and that cancers develop in tissues that harbor normal-LRC, we propose that LRCC might represent a novel population of GI stem-like cancer cells. LRCC may provide novel mechanistic insights into the biology of cancer and regenerative medicine and present novel targets for cancer treatment.

Liver Label Retaining Cancer Cells Are Relatively Resistant to the Reported Anti-Cancer Stem Cell Drug Metformin.

BACKGROUND & AIMS: Recently, we reported that liver Label Retaining Cancer Cells (LRCC) can initiate tumors with only 10 cells and are relatively resistant to the targeted drug Sorafenib, a standard of practice in advanced hepatocellular carcinoma (HCC). LRCC are the only cancer stem cells (CSC) isolated alive according to a stem cell fundamental function, asymmetric cell division. Metformin has been reported to preferentially target many other types of CSC of different organs, including liver. It's important to know if LRCC, a novel class of CSC, are relatively resistant to metformin, unlike other types of CSC. As metformin inhibits the Sorafenib-Target-Protein (STP) PI3K, and LRCC are newly described CSC, we undertook this study to test the effects of Metformin on Sorafenib-treated HCC and HCC-derived-LRCC. METHODS: We tested various STP levels and phosphorylation status, associated genes' expression, proliferation, viability, toxicity, and apoptosis profiles, before and after treatment with Sorafenib with/without Metformin. RESULTS: Metformin enhances the effects of Sorafenib on HCC, and significantly decreased viability/proliferation of HCC cells. This insulin-independent effect was associated with inhibition of multiple STPs (PKC, ERK, JNK and AKT). However, Metformin increased the relative proportion of LRCCs. Comparing LRCC vs. non-LRCC, this effect was associated with improved toxicity and apoptosis profiles, down-regulation of cell death genes and up-regulation of cell proliferation and survival genes in LRCC. Concomitantly, Metformin up-regulated pluripotency, Wnt, Notch and SHH pathways genes in LRCC vs. non-LRCC. CONCLUSIONS: Metformin and Sorafenib have enhanced anti-cancer effects. However, in contradistinction to reports on other types of CSC, Metformin is less effective against HCC-derived-CSC LRCC. Our results suggest that combining Metformin with Sorafenib may be able to repress the bulk of tumor cells, but as with other anti-cancer drugs, may leave LRCC behind leading to cancer recurrence. Therefore, liver LRCC, unlike other types of CSC, are relatively resistant to the reported anti-cancer stem cell drug metformin. This is the first report that there is a type of CSC that is not relatively resistant to the CSC-targeting drug. Our findings suggest that a drug targeting LRCC may be critically needed to target CSC and prevent cancer recurrence. These may significantly contribute to the understanding of Metformin's anti-cancer effects and the development of novel drugs targeting the relatively resistant LRCC.

Wnt and the cancer niche: paracrine interactions with gastrointestinal cancer cells undergoing asymmetric cell division.

OBJECTIVE: Stem-like cancer cells contribute to cancer initiation and maintenance. Stem cells can self-renew by asymmetric cell division (ACD). ACD with non-random chromosomal cosegregation (ACD-NRCC) is one possible self-renewal mechanism. There is a paucity of evidence supporting ACD-NRCC in human cancer. Our aim was to investigate ACD-NRCC and its potential interactions with the cancer niche (microenvironment) in gastrointestinal cancers. DESIGN: We used DNA double and single labeling approaches with FACS to isolate live cells undergoing ACD-NRCC. RESULTS: Gastrointestinal cancers contain rare subpopulations of cells capable of ACD-NRCC. ACD-NRCC was detected preferentially in subpopulations of cells previously suggested to be stem-like/tumor-initiating cancer cells. ACD-NRCC was independent of cell-to-cell contact, and was regulated by the cancer niche in a heat-sensitive paracrine fashion. Wnt pathway genes and proteins are differentially expressed in cells undergoing ACD-NRCC vs. symmetric cell division. Blocking the Wnt pathway with IWP2 (WNT antagonist) or siRNA-TCF4 resulted in suppression of ACD-NRCC. However, using a Wnt-agonist did not increase the relative proportion of cells undergoing ACD-NRCC. CONCLUSION: Gastrointestinal cancers contain subpopulations of cells capable of ACD-NRCC. Here we show for the first time that ACD-NRCC can be regulated by the Wnt pathway, and by the cancer niche in a paracrine fashion. However, whether ACD-NRCC is exclusively associated with stem-like cancer cells remains to be determined. Further study of these findings might generate novel insights into stem cell and cancer biology. Targeting the mechanism of ACD-NRCC might engender novel approaches for cancer therapy.

Comparative testing of various pancreatic cancer stem cells results in a novel class of pancreatic-cancer-initiating cells.

No systemic therapy is effective against pancreatic cancer (PC). Pancreatic cancer stem cells (PCSC) are hypothesized to account for therapeutic resistance. Several PCSC subpopulations were reported, each characterized by different markers. To be able to target PCSC, we sought to better define this putative heterogeneity. Therefore, we tested most of the known putative PCSC markers in established and fresh tumor cell lines. CD20, CD24, CD44, CD133, CD184 (CXCR4), CD326 (EpCam, ESA), Sox-2, OCT 3/4, and the side-population (SP) were tested in five PC cell lines, and the effects of confluency, hypoxia, radiation, and gemcitabine on the SP. The testing phase suggested several putative PCSC populations that were further tested and validated for their tumor-initiating capacity against known PCSC in 3 established and 1 fresh PC cell lines. Cell surface and intracellular markers showed significant variability among cell lines. SP was the only common marker in all cell lines and consistently less than 1%. SP response to confluence, hypoxia, radiation, and gemcitabine was inconsistent between cell lines. The initial testing phase suggested that SP/CD44-CD24-CD326+ cells might be a novel PCSC subpopulation. Tumor initiation capacity tests in nude mice confirmed their increased tumorigenicity over previously reported PCSC. Our data better define the heterogeneity of reported PCSC in cell lines tested in this study. We propose that prior to targeting PC via PCSC, one will need to gain more insight into this heterogeneity. Finally, we show that SP/CD44-CD24-CD326+ cells are a novel subpopulation of pancreatic cancer tumor initiating cells. Further mechanistic studies may lead to better targeting of PC via targeting this novel PCSC.

Establishment of human ultra-low passage colorectal cancer cell lines using spheroids from fresh surgical specimens suitable for in vitro and in vivo studies.

Colorectal cancer (CRC) holds the third highest incidence and cancer related mortality rate among men and women in the United States. Unfortunately, there has been little progression made in the treatment of this deadly disease once it has spread beyond the colon. It has been hypothesize that colon cancer stem cells are implicated in CRC carcinogenesis, metastasis, and therapeutic resistance. One of the difficulties in testing these hypotheses is the current use of established high-passage cancer cell lines. Long term, high-passage established cell lines have cells with stem like properties as they propagate almost indefinitely. These cells are thought to be different than the original cancer stem cells in fresh tumors. In order to investigate cancer stem cells, and molecularly profiling tumors with high fidelity to the original primary tumor, one needs to establish suitable primary ultra-low passage cell lines from fresh surgical specimens. Here we report the establishment of tumor initiating colon cancer ultra-low passage cell lines by a combination of gentle mechanical, enzymatic dissociation, spheroid formation, and followed by two generation xenografts from fresh tumors obtained at time of operation. Tumors generated were characterized by morphology, flow cytometry, immunofluorescence, and by gene expression. In the future, such a technology can be used to produce expeditiously enough material to test for mutations, genetic signatures and molecular subtyping readily available for clinical therapeutic decision making.

A Pilot Study Assessing the Potential Role of non-CD133 Colorectal Cancer Stem Cells as Biomarkers.

INTRODUCTION: Over 50% of patients with colorectal cancer (CRC) will progress and/or develop metastases. Biomarkers capable of predicting progression, risk stratification and therapeutic benefit are needed. Cancer stem cells are thought to be responsible for tumor initiation, dissemination and treatment failure. Therefore, we hypothesized that CRC cancer stem cell markers (CRCSC) will identify a group of patients at high risk for progression. METHODS: Paraffin-embedded tissue cores of normal (n=8), and histopathologically well-defined primary (n= 30) and metastatic (n=10) CRC were arrayed in duplicate on tissue microarrays (TMAs). Expression profiles of non-CD133 CRCSC (CD29, CD44, ALDH1A1, ALDH1B1, EpCam, and CD166) were detected by immunohistochemistry and the association with clinicopathological data and patient outcomes was determined using standard statistical methodology. An independent pathologist, blinded to the clinical data scored the samples. Scoring included percent positive cells (0 to 4, 0 = <10%, 1 = 10 - 24%, 2 = 25 - 49%, 3 = 50 - 74%, 4 = 75 - 100%), and the intensity of positively stained cells (0 to 4; 0 = no staining, 1 = diminutive intensity, 2 = low intensity, 3 = intermediate intensity, 4 = high intensity). The pathologic score represents the sum of these two values, reported in this paper as a combined IHC staining score (CSS). RESULTS: Of 30 patients 7 were AJCC stage IIA, 10 stage IIIB, 7 stage IIIC and 6 stage IV. Median follow-up was 113 months. DFI was 17 months. Median overall survival (OS) was not reached. Stage-specific OS was: II - not reached; III - not reached; IV - 11 months. In a univariate analysis, poor OS was associated with loss of CD29 expression; median OS, 32 months vs. not reached for CSS 3-7 vs. >7.5, respectively; p=0.052 comparing entire curves, after adjustment. In a Cox model analysis, loss of CD29 exhibited a trend toward association with survival (p=0.098) after adjusting for the effect of stage (p=0.0076). Greater expression of ALDH1A1 was associated with increasing stage (p=0.042 over stages 2, 3b, 3c, and 4) while loss of CD29 expression exhibited a trend toward being associated with stages 3 and 4 (p=0.08). Compared to normal colon tissue, primary tumors were associated with increased expression of ALDH1B1 (p=0.008). ALD1H1B1 expression level differed according to whether the tumor was moderately or poorly differentiated, well differentiated, or mucinous; the highest expression levels were associated with moderately or poorly differentiated tumors (p=0.011). Lymph node metastases were associated with a trend toward decreased expression of EpCAM (p = 0.06) when comparing 0 vs. 1 vs. 2+ positive lymph nodes, as was CD29 (p = 0.08) when comparing 0 vs. any positive lymph nodes. Compared to normal colon tissue metastatic colon cancers from different patients were associated with increased ALDH1B1 expression (p=0.001) whereas CD29 expression was higher in normal colonic tissue (p=0.014). CONCLUSION: CD29 may be associated with survival as well as clinical stage and number of lymph nodes. ALDH1B1 expression was associated with differentiation as well as type of tissue evaluated. ALDH1A1 was associated with clinical stage, and decreased EpCAM expression was found in patients with advanced lymph node stage. CRCSCs may be useful biomarkers to risk stratify, and estimate outcomes in CRC. Larger prospective studies are required to validate the current findings.

Mithramycin represses basal and cigarette smoke-induced expression of ABCG2 and inhibits stem cell signaling in lung and esophageal cancer cells.

Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

Isolation of live label-retaining cells and cells undergoing asymmetric cell division via nonrandom chromosomal cosegregation from human cancers.

The ability to retain DNA labels over time is a property proposed to be associated with adult stem cells. Recently, label retaining cells (LRC) were indentified in cancer. LRC were suggested to be the result of either slow-cycling or asymmetric-cell-division with nonrandom-chromosomal-cosegregation (ACD-NRCC). ACD-NRCC is proposed to segregate the older template DNA strands into daughter stem cells and newly synthesized DNA into daughter cells destined for differentiation. The existence of cells undergoing ACD-NRCC and the stem-like nature of LRC remain controversial. Currently, to detect LRC and ACD-NRCC, cells need to undergo fixation. Therefore, testing the stem-cell nature and other functional traits of LRC and cells undergoing ACD-NRCC has been limited. Here, we show a method for labeling DNA with single and dual-color nucleotides in live human liver cancer cells avoiding the need for fixation. We describe a novel methodology for both the isolation of live LRC and cells undergoing ACD-NRCC via fluorescence-activated cell sorting with confocal microscopy validation. This has the potential to be a powerful adjunct to stem-cell and cancer research.

A functional mouse retroposed gene Rps23r1 reduces Alzheimer's beta-amyloid levels and tau phosphorylation.

Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimer's disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.