Deniz A. Ucar

Aldehyde dehydrogenase activity as a functional marker for lung cancer

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore, the ALDH(br) cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion, ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.

The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance.

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.

Bone Marrow Contributes to Epithelial Cancers in Mice and Humans as Developmental Mimicry

Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry.

Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion.

LKB1 is a tumor susceptibility gene for the Peutz–Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1–CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1–CRTC signaling might represent a crucial mechanism for esophageal cancer progression.

Targeting of the protein interaction site between FAK and IGF-1R

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth.

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.

Inhibiting the Interaction of cMET and IGF-1R with FAK Effectively Reduces Growth of Pancreatic Cancer Cells in vitro and in vivo

Pancreatic cancer is one of the most lethal diseases with no effective treatment. Previously, we have shown that FAK is overexpressed in pancreatic cancer and plays a key role in cancer cell survival and proliferation. FAK has been shown to interact with growth factor receptors including cMET and IGF-1R. As a novel therapeutic approach, we targeted the protein interaction of FAK with growth factor receptors to block tumor growth, alter signaling pathways and sensitize cells to chemotherapy. We have selected a small molecule compound (INT2-31) that decreases phosphorylation of AKT via disrupting interaction of FAK with cMET and IGF-1R. Our results demonstrate that interaction of a small molecule compound with FAK decreases phosphorylation of FAK Y397 while increasing FAK Y407 phosphorylation, without inhibiting the kinase activity of FAK and dramatically reduces downstream signaling to AKT. Our lead compound, INT2-31, demonstrates significant inhibition of tumor cell growth in two orthotopic models of pancreatic cancer. In addition, INT2-31 increases sensitivity to gemcitabine chemotherapy in a direct fresh biopsy xenograft model of pancreatic cancer growth.

FAK and Interacting Proteins as Therapeutic Targets in Pancreatic Cancer

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Chemotherapy and radiation therapy have had minimal ability to alter the natural course of the disease. Clearly, additional agents are needed to improve outcomes in this aggressive cancer. Pancreatic cancer has been found to have several genetic alterations including activation of K-ras and inactivation of p53, p16, and DPC4. Other alterations include upregulation of angiogenic factors and matrix metalloproteinases, dysregulation of growth factor receptors, and cytoplasmic kinases including focal adhesion kinase (FAK) and src. Clinicians must translate the available knowledge of the molecular basis of this disease into rationale and effective therapeutic strategies for treatment. The role of FAK in the pathogenesis of pancreatic cancer is discussed below and efforts aimed at the development of inhibitors of FAK for this disease are reviewed.

A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells

Melanoma has the highest mortality rate of all skin cancers and a major cause of treatment failure is drug resistance. Tumors heterogeneity requires novel therapeutic strategies and new drugs targeting multiple pathways. One of the new approaches is targeting the scaffolding function of tumor related proteins such as focal adhesion kinase (FAK). FAK is overexpressed in most solid tumors and is involved in multiple protein-protein interactions critical for tumor cell survival, tumor neovascularization, progression and metastasis. In this study, we investigated the anticancer activity of the FAK scaffold inhibitor C4, targeted to the FAK-VEGFR-3 complex, against melanomas. We compared C4 inhibitory effects in BRAF mutant vs BRAF wild type melanomas. C4 effectively caused melanoma tumor regression in vivo, when administered alone and sensitized tumors to chemotherapy. The most dramatic effect of C4 was related to reduction of vasculature of both BRAF wild type and V600E mutant xenograft tumors. The in vivo effects of C4 were assessed in xenograft models using non-invasive multimodality imaging in conjunction with histologic and molecular biology methods. C4 inhibited cell viability, adhesion and motility of melanoma and endothelial cells, specifically blocked phosphorylation of VEGFR-3 and FAK and disrupted their complexes. Specificity of in vivo effects for C4 were confirmed by a decrease in tumor FAK and VEGFR-3 phosphorylation, reduction of vasculogenesis and reduced blood flow. Our collective observations provide evidence that a small molecule inhibitor targeted to the FAK protein-protein interaction site successfully inhibits melanoma growth through dual targeting of tumor and endothelial cells and is effective against both BRAF wild type and mutant melanomas.

A novel small molecule inhibitor of FAK and IGF-1R protein interactions decreases growth of human esophageal carcinoma.

INTRODUCTION Esophageal cancer remains an aggressive disease with poor survival rates. FAK and IGF-1R are two important tyrosine kinases important for cell survival signaling and found to be upregulated in esophageal cancer. Our hypothesis is that a novel small molecule compound that disrupts FAK and IGF-1R protein-protein interactions (PPIs) would decrease the growth of human esophageal cancer. METHODS The compound INT2-31 (NSC344553) was identified from a virtual high throughput screen to bind to FAK and disrupt PPIs. The in vitro effects of this compound, +/- 5-FU chemotherapy, on cell signaling, viability and apoptosis in human esophageal cancer cells (KYSE 70, 140) and a direct esophageal cancer xenograft was evaluated. RESULTS INT2-31 caused a disruption of PPIs between FAK and IGF-1R starting at a concentration of 1μM. It also caused a dose dependent inhibition of cell viability and induction of apoptosis at low micromolar doses. These effects were associated with decreased AKT and ERK1/ERK2 phosphorylation. INT2-31 treatment, when administered via IP injection, at 50mg/kg, resulted in an in vivo decrease in tumor growth in a direct xenograft. Furthermore, treatment with 5-FU chemotherapy combined with INT2-31 resulted in a synergistic increase in apoptosis and decrease in tumor growth compared to 5-FU or INT2-31 alone. CONCLUSIONS A novel compound that disrupts the PPIs of FAK and IGF-1R results in decreased tumor proliferation and increased apoptosis. These effects appear to be mediated through downregulation of p-AKT and p-ERK. This compound deserves further study as a novel treatment strategy in patients with esophageal cancer.