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Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.

Pertuzumab is FDA approved in the preoperative setting in combination with trastuzumab and chemotherapy, in women with nonmetastatic HER2 + breast cancer. The TRYPHAENA trial (n = 77) reported a pathologic complete response rate (pCR), i.e., ypT0ypN0, of 52 % in patients treated with neoadjuvant (docetaxel, carboplatin, trastuzumab,pertuzumab) TCH-P. Aside from this study, there is limited information regarding the safety and efficacy of TCH-P in the neoadjuvant setting. Our goal was to evaluate the safety and efficacy of neoadjuvant TCH-P in a non-clinical trial setting.Cancer data registry was utilized to identify patients with HER2 + nonmetastatic breast cancer that received neoadjuvant TCH-P. pCR was defined as the absence of invasive or noninvasive cancer in breast and lymph nodes, i.e., ypT0ypN0.70 patients with a median age of 52 years met our inclusion criteria. Clinical staging was I-8.5 %; II-68.5 %; and III-22.8 %. 60 % of patients had hormone receptor (HR)-positive tumors. 23 % (16/71) of patients required dose reduction for rash, diarrhea, neuropathy, or thrombocytopenia. Overall, no patients developed grade 3-4 left ventricular systolic dysfunction(LVSD); an asymptomatic reduction in LVEF of 10 % was observed in three patients. The overall observed pCR rate was 53 %. As expected, the pCR rate was higher in patients with HR-negative breast cancer than for patients with HR+ disease: 69 % (20/29) vs. 42 % (17/41), respectively. The axillary downstaging rate was approximately 53 % (19/36).Neoadjuvant TCH-P, in a nonclinical trial setting, was associated with a pCR rate of 53 % similar the reported rate in TRYPHAENA. Toxicity was manageable, with no patients experiencing symptomatic heart failure.

5-Fluorouracil (5-FU) continues to be widely used for treatment of gastrointestinal cancers. Because many tumors show primary or acquired resistance, it is important to understand the molecular basis underlying the mechanism of resistance to 5-FU. In addition to its effect on thymidylate synthase inhibition and DNA synthesis, 5-FU may also influence RNA metabolism. Our previous studies revealed that colorectal cancer cells resistant to bolus 5-FU (HCT-8/4hFU) showed significantly decreased incorporation of the drug into RNA. Resistance to bolus 5-FU was associated with lower expression of UMP kinase (UMPK), an enzyme that plays an important role in the activation of 5-FU to 5-FUTP and its incorporation into RNA. Activities of other 5-FU–metabolizing enzymes (e.g., thymidine kinase, uridine phosphorylase, thymidine phosphorylase, and orotate phosphoribosyltransferase) remained unchanged between sensitive and resistant cell lines. Herein, we show that UMPK down-regulation in 5-FU–sensitive cells (HCT-8/P) induces resistance to bolus 5-FU treatment. Moreover, HCT-8/4hFU cells are even more cross-resistant to treatment with 5-fluorouridine, consistent with the current understanding of 5-fluorouridine as a RNA-directed drug. Importantly, colorectal cancer hepatic metastases isolated from patients clinically resistant to weekly bolus 5-FU/leucovorin treatment exhibited decreased mRNA expression of UMPK but not thymidylate synthase or dihydropyrimidine dehydrogenase compared with tumor samples of patients not previously exposed to 5-FU. Our findings provide new insights into the mechanisms of acquired resistance to 5-FU in colorectal cancer and implicate UMPK as an important mechanism of clinical resistance to pulse 5-FU treatment in some patients.[Mol Cancer Ther 2009;8(4):OF1–8]

a Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA b Graduate Program in Molecular Medicine, Boston University School of Medicine, Boston, MA 02215, USA c Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM–derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

AIM: To compare the efficacy of cell-free derivatives from Bone marrow derived human mesenchymal stem cells (hMSCs) in wound therapy. METHODS: hMSCs have been shown to play an important role in wound therapy. The present study sought to compare efficacy of hMSCs and cell-free derivatives of hMSCs, which may be clinically more relevant as they are easier to prepare, formulate and transport. hMSCs were isolated from human bone marrow and cultured. Multi lineage differentiation of hMSCs was performed to confirm their identity. The ability of hMSCs to migrate was evaluated using in vitro and in vivo migration assays. Cell lysates and conditioned medium concentrate was prepared from hMSCs (see Methods for details). Wounds were induced in mice and wound areas were measure before and after cell and cell-free derivative treatment. RNA and proteins were extracted from the skin and cytokine levels were measured. RESULTS: Co-culture of hMSCs with keratinocytes resulted in increased expression of CXCL-12 (SDF1) and ENA78 (CXCL-5) in the conditioned media indicating that the hMSCs can respond to signals from keratinocytes. Accelerated wound closure was observed when hMSCs were injected near the site of excisional wounds in athymic as well as NOD/SCID mice. Interestingly, cell-free lysates prepared from hMSCs were also effective in inducing accelerated wound closure and increased expression of SDF1 and CXCL-5 at the wound bed. Additionally, concentrated media from hMSCs as well as an emulsion containing lysates prepared from hMSCs was also found to be more effective in rapid re-epithelialization than fibroblasts or vehicle-alone control. Use of cell-free derivatives may help replace expensive wound care approaches including use of growth factors, epidermal/dermal substitutes, synthetic membranes, cytokines, and matrix components, and most importantly avoid transmission of pathogens from human and animal products. CONCLUSION: These results encourage development of derivatives of hMSCs for wound care and re-epithelialization applications.

Tumor-associated fibroblasts or carcinoma-associated fibroblasts (CAF) play an important role in the growth of epithelial solid tumors. Although the cell type of origin of CAFs has not been conclusively established, it has been shown that they may be bone marrow derived. One side of the mesenchymal stem cell (MSC) coin is the well-accepted therapeutic potential of these cells for regenerative and immunomodulatory purposes. The ominous dark side is revealed by the recent work demonstrating that hMSCs may be a source of CAFs. In this review, we discuss the role of stromal cells in the tumor microenvironment and suggest that by exploring the in vitro/in vivo interplay between different cell types within the tumor milieu, strategies for improved tumor therapy can be developed. [Cancer Res 2009;69(4):1255–8]

MSCs are adult multipotent stromal cells with the potential to differentiate into a variety of cell types. They are able to migrate toward an injury site and when there become bone, cartilage, fat or muscle, repairing or replacing the damaged connective tissue; however, MSCs can also be recruited to tumor sites

Joseph R Bertino1†, Debabrata Banerjee2 & Prasun J Mishra3 †Author for correspondence 1The Cancer Institute of New Jersey, Department of Medicine and Pharmacology, UMDNJ-Robert Wood Johnson Medical School, 195, Little Albany Street, Room 3033, New Brunswick, NJ 08903, USA Tel.: +1 732 235 8510; Fax: +1 732 235 8181; E-mail: bertinoj@umdnj.edu 2GSBS, Department of Pharmacology, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, UMDNJ, New Brunswick, NJ 08903, USA 3Department of Pharmacology and Medicine, Cancer Institute of New Jersey, Robert Wood Johnson Medical School, UMDNJ, New Brunswick, NJ 08903, USA ‘The understanding of how an individual’s genetic inheritance of miRSNPs/polymorphisms affects the body’s response to certain drugs will be key to creating drugs with greater efficacy and safety.’

Mesenchymal stem cells (MSCs) are multipotent cells with a number of potential therapeutic applications. At present, they are being used in a clinical trial for the treatment of myocardial infarction and are being studied as a therapy for other vascular disorders. Treatments of neurologic disorders and anticancer therapy with MSCs have progressed in light of the migratory properties of MSCs to brain injury and tumors. The osteogenic potential of MSCs is being exploited in work investigating their use in bone regeneration therapy, and the immunomodulatory function of MSCs is being evaluated as a possible therapy for graft-versus-host disease. Here, the authors review recent work contributing to the knowledge of MSC biology and the advances in gene therapy and tissue regeneration using MSCs.

Metformin is a biguanine derivative, widely used as an antidiabetic drug. Epidemiological studies on several types of cancers including breast, prostate, ovarian and pancreatic cancer show that patients treated with metformin have a lower incidence of cancer. Mechanistic studies have shown that metformin inhibits cancer cell viability by activating the AMP activated protein kinase (AMPK) pathway and inhibiting mTOR. In this study we developed a metformin resistance LnCap prostate cancer cells by exposing the cells continuously increasing concentrations of metformin. Micro-array data analysis comparing the resistant cells (4-fold) to parental cells showed that genes associated with receptor binding and with cell proliferation are primarily affected. EDIL3 (EGF-like repeats and discoidin I-like domains 3), EREG (eEpiregulin) and AXL (Axl receptor tyrosine kinase) are top three up regulated genes (∼ 35 fold). CALB2 (cCalbindin 2) and TPTE (transmembrane phosphatase with tensin homology) were the top down regulated genes (10 fold down regulated). AXL overexpression has earlier been demonstrated in Imatinib resistant CML cell lines and in CML cells from patients. We are currently investigating the role of Axl receptor tyrosine kinase in metformin resistant prostate cancer cells. Citation Format: Nitu Bansal, Prasun Mishra, Kathleen Cruz, Joseph Bertino. Receptor tyrosine kinase AXL is upregulated in metformin resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 996. doi:10.1158/1538-7445.AM2013-996

Carcinoma Associated Fibroblasts (CAFs) along with other cells present in the tumor milieu play a major role in tumor progression. We have previously demonstrated differentiation of human bone marrow derived mesenchymal stem cells to CAF like phenotype upon exposure to tumor conditioned medium. The present study evaluates the ability of these stromal components to support tumor progression. An in vitro system was used to study impact of the components of the tumor milieu on growth of luciferase expressing MDA MB 231 cells, representing basal subtype of human breast cancer. CAFs along with phorbol ester induced HL60 cells representing a macrophage like phenotype supported growth of MDAMB231 cells in vitro as measured by a luciferase assay. When injected subcutaneously in vivo in nu/nu mice, tumor cells comingled with CAFs or with differentiated HL60 cells showed almost similar tumor growth rate, whereas CAFs and differentiated HL60s when mixed together showed more than 4 fold increased tumor growth rate compared to the tumor cells alone and 2 fold compared to tumor cells plus MatrigelTM. Electrophoretic analysis of extracellular matrix protein mixture isolated from mixed cultures of CAFs plus MDAMB231 cells and differentiated HL60 cells (3 cells) using previously published methods showed presence of laminins and collagens, and demonstrated robust tumor cell growth compared to MatrigelTM. The ECM protein mixture derived from the three cell system offers a completely humanized substitute for MatrigelTM useful for study of tumor growth and response to therapy in vitro as well as in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 556.