Bikul Das

Hypoxia Enhances Tumor Stemness by Increasing the Invasive and Tumorigenic Side Population Fraction

Although advances have been made in understanding the role of hypoxia in the stem cell niche, almost nothing is known about a potentially similar role of hypoxia in maintaining the tumor stem cell (TSC) niche. Here we show that a highly tumorigenic fraction of side population (SP) cells is localized in the hypoxic zones of solid tumors in vivo. We first identified a highly migratory, invasive, and tumorigenic fraction of post‐hypoxic side population cells (SPm[hox] fraction) in a diverse group of solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, and small‐cell lung carcinoma. To identify the SPm(hox) fraction, we used an “injured conditioned medium” derived from bone marrow stromal cells treated with hypoxia and oxidative stress. We found that a highly tumorigenic SP fraction migrates to the injured conditioned medium in a Boyden chamber. We show that as few as 100 SPm(hox) cells form rapidly growing tumors in vivo. In vitro exposure to hypoxia increases the SPm(hox) fraction significantly. Quantitative real‐time polymerase chain reaction and immunofluorescence studies showed that SPm(hox) cells expressed Oct‐4, a “stemness” gene having a potential role in TSC maintenance. In nude mice xenografts, SPm(hox) cells were localized to the hypoxic zones, as demonstrated after quantum dot labeling. These results suggest that a highly tumorigenic SP fraction migrates to the area of hypoxia; this migration is similar to the migration of normal bone marrow SP fraction to the area of injury/hypoxia. Furthermore, the hypoxic microenvironment may serve as a niche for the highly tumorigenic fraction of SP cells.

Vascular endothelial growth factor acts in an autocrine manner in rhabdomyosarcoma cell lines and can be inhibited with all-trans-retinoic acid

Vascular endothelial growth factor (VEGF) is a potent signalling molecule that acts through two tyrosine kinase receptors, VEGFR1 and VEGFR2. The upregulation of VEGF and its receptors is important in tumour-associated angiogenesis; however, recent studies suggest that several tumour cells express VEGF receptors and may be influenced by autocrine VEGF signalling. Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma, and is dependent on autocrine signalling for its growth. The alveolar subtype of RMS is often characterized by the presence of a PAX3-FKHR translocation, and when introduced into non-RMS cells, the resultant fusion protein induces expression of VEGFR1. In our study, we examined the expression of VEGF and its receptors in RMS, and autocrine effects of VEGF on cell growth. VEGF and receptor mRNA and protein were found to be expressed in RMS cells. Exogenous VEGF addition resulted in extracellular signal-regulated kinase-1/2 phosphorylation and cell proliferation, and both were reduced by VEGFR1 blockade. Growth was also slowed by VEGFR1 inhibitor alone. Treatment of RMS cells with all-trans-retinoic acid decreased VEGF secretion and slowed cell growth, which was rescued by VEGF. These data suggest that autocrine VEGF signalling likely influences RMS growth and its inhibition may be an effective treatment for RMS.

Cisplatin treatment increases survival and expansion of a highly tumorigenic side-population fraction by upregulating VEGF/Flt1 autocrine signaling

The cellular and molecular mechanisms of tumor progression following chemotherapy are largely unknown. Here, we demonstrate that cisplatin (CDDP) treatment upregulates VEGF and Flt1 expression leading to the survival and expansion of a highly tumorigenic fraction of side-population (SP) cells in osteosarcoma (HOS), neuroblastoma (SK-N-BE2) and rhabdomyosarcoma (RH-4) cell lines. In all three lines, we show that CDDP treatment increases levels of VEGF and Flt1 expression, and induces enhanced clonogenic capacity and increased expression of the ‘stemness’-associated genes Nanog, Bmi-1 and Oct-4 in the SP fraction. In HOS, these changes are associated with the transformation of a non-tumorigenic osteosarcoma SP fraction to a highly tumorigenic phenotype. Inhibition of Flt1 led to complete reduction of tumorigenicity in the HOS SP fraction, and reduction of clonogenic capacity and expression of stemness genes in the SK-N-BE(2) and RH-4 SP fractions. Treatment with U0126, a specific inhibitor of MAPK/ERK1,2 completely downregulates CDDP-induced VEGF and Flt1 expression and induction/expansion of SP fraction in all three cell lines, indicating that these effects are mediated through MAPK/ERK1,2 signaling. In conclusion, we report a novel mechanism of CDDP-induced tumor progression, whereby the activation of VEGF/Flt1 autocrine signaling leads to the survival and expansion of a highly tumorigenic SP fraction.

Combination therapy in combating cancer

Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-cancer drugs enhances efficacy compared to the mono-therapy approach because it targets key pathways in a characteristically synergistic or an additive manner. This approach potentially reduces drug resistance, while simultaneously providing therapeutic anti-cancer benefits, such as reducing tumour growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. The 5-year survival rates for most metastatic cancers are still quite low, and the process of developing a new anti-cancer drug is costly and extremely time-consuming. Therefore, new strategies that target the survival pathways that provide efficient and effective results at an affordable cost are being considered. One such approach incorporates repurposing therapeutic agents initially used for the treatment of different diseases other than cancer. This approach is effective primarily when the FDA-approved agent targets similar pathways found in cancer. Because one of the drugs used in combination therapy is already FDA-approved, overall costs of combination therapy research are reduced. This increases cost efficiency of therapy, thereby benefiting the “medically underserved”. In addition, an approach that combines repurposed pharmaceutical agents with other therapeutics has shown promising results in mitigating tumour burden. In this systematic review, we discuss important pathways commonly targeted in cancer therapy. Furthermore, we also review important repurposed or primary anti-cancer agents that have gained popularity in clinical trials and research since 2012.

HIF-2α Suppresses p53 to Enhance the Stemness and Regenerative Potential of Human Embryonic Stem Cells†‡§

Human embryonic stem cells (hESCs) have been reported to exert cytoprotective activity in the area of tissue injury. However, hypoxia/oxidative stress prevailing in the area of injury could activate p53, leading to death and differentiation of hESCs. Here we report that when exposed to hypoxia/oxidative stress, a small fraction of hESCs, namely the SSEA3+/ABCG2+ fraction undergoes a transient state of reprogramming to a low p53 and high hypoxia inducible factor (HIF)‐2α state of transcriptional activity. This state can be sustained for a period of 2 weeks and is associated with enhanced transcriptional activity of Oct‐4 and Nanog, concomitant with high teratomagenic potential. Conditioned medium obtained from the post‐hypoxia SSEA3+/ABCG2+ hESCs showed cytoprotection both in vitro and in vivo. We termed this phenotype as the “enhanced stemness” state. We then demonstrated that the underlying molecular mechanism of this transient phenotype of enhanced stemness involved high Bcl‐2, fibroblast growth factor (FGF)‐2, and MDM2 expression and an altered state of the p53/MDM2 oscillation system. Specific silencing of HIF‐2α and p53 resisted the reprogramming of SSEA3+/ABCG2+ to the enhanced stemness phenotype. Thus, our studies have uncovered a unique transient reprogramming activity in hESCs, the enhanced stemness reprogramming where a highly cytoprotective and undifferentiated state is achieved by transiently suppressing p53 activity. We suggest that this transient reprogramming is a form of stem cell altruism that benefits the surrounding tissues during the process of tissue regeneration. STEM CELLS2012;30:1685–1695

SBDS‐deficiency results in deregulation of reactive oxygen species leading to increased cell death and decreased cell growth

Shwachman–Diamond syndrome (SDS) is characterized by reduced hematopoietic and exocrine pancreatic cell numbers and a marked propensity for leukemia. Most patients have mutations in the SBDS gene. We previously reported that SBDS‐deficient cells overexpress Fas, undergo accelerated spontaneous and Fas‐mediated apoptosis and grow slowly. However the mechanism of how SBDS regulates apoptosis remains unknown. Several studies have shown that reactive oxygen species (ROS) regulate cell growth and spontaneous and Fas‐mediated cell death. Therefore, we hypothesized that SBDS‐deficiency disrupts ROS regulation and subsequently increases sensitivity to Fas stimulation and reduced cell growth.

Meta-[123I]iodobenzylguanidine is selectively radiotoxic to neuroblastoma cells at concentrations that spare cells of haematopoietic lineage.

BackgroundThe Auger electron-emitting agents meta-[125I]iodobenzylguanidine (125I-MIBG) and 123I-MIBG have been proposed as alternatives to 131I-MIBG for the treatment of neuroblastoma, due to the absence of a cross-fire effect which may minimize bone marrow toxicity. However, the differential toxicity of 123I-MIBG towards neuroblastoma cells and cells of haematopoietic lineage has not been studied. ObjectiveTo compare the toxic effects of 123I-MIBG on SK-N-SH and SK-N-BE(2) neuroblastoma cells and on cells of haematopoietic lineage, specifically HL-60 human myeloid leukemia cells and bone marrow stem cells (BMSCs) from human adult donors. MethodsThe antiproliferative effects of exchange-labelled or no carrier added (n.c.a.) 123I-MIBG, unlabelled MIBG or the trimethylsilylbenzylguanidine (MTBG) precursor used to prepare n.c.a. 123I-MIBG against SK-N-SH or SK-N-BE(2) cells or HL-60 cells were evaluated using a cell proliferation assay. The toxicity of 123I-MIBG towards SK-N-SH cells or BMSCs from healthy adult human donors was studied using a clonogenic assay. Results123I-MIBG was strongly growth inhibitory to SK-N-SH or SK-N-BE(2) cells at concentrations (IC50 185-370 mBq·ml−1; IC90 740 mBq·ml−1) that were sparing to HL-60 cells. Treatment of SK-N-SH cells with 74 mBq of 123I-MIBG decreased colony formation by >90%, whereas colonies from all three populations of stem cells were formed at amounts up to 370 mBq. It was discovered that the MTBG precursor was non-specifically toxic towards both SK-N-SH cells and HL-60 cells, suggesting the need to purify n.c.a. 123I-MIBG for clinical use. ConclusionOur results suggest that 123I-MIBG is a promising novel radiotherapeutic agent for neuroblastoma. For the first time, we report that the MTBG precursor used to prepare n.c.a. 123I-MIBG was toxic towards neuroblastoma cells as well as to HL-60 cells, representing cells of the haematopoietic lineage, suggesting the need for purification.

The Idea and Evidence for the Tumor Stemness Switch

The maintenance of stemness of normal stem cell is a complex process, where transcription factors like Oct-4, Bmi-1, and signaling pathways such as Wnt/β-catenin play important roles. This molecular set of mechanisms not only expands the population (self-renewal) but also keeps stem cells in a state of “de-differentiation.” Thus, stemness and differentiation are mutually exclusive and tightly regulated, where the idea of variation of stemness over time has not been incorporated. However, unlike normal stem cell stemness, tumor stemness may not be tightly regulated, where complexity of tumor microenvironment, especially hypoxic stress may allow for variation in stemness.

Abstract 3903: Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells

Background: Tumor hypoxia is a major contributing factor in cancer therapeutic failure. The microenvironment of hypoxia/oxidative stress may reprogram cancer cells to highly tumorigenic, metastatic, angiogenic and stem cell-like state. We have characterized in vitro and in vivo models of hypoxia-induced cancer cell reprogramming to cancer stem cell (CSCs) like cells including an immunocompetent model of oral cancer. These CSCs exhibit ABCG2 cell surface marker. Numerous approaches including targeted therapy using oncolytic virus and bacteria have been attempted to target cancer cells in their hypoxic niche without major therapeutic success. Major therapeutic challenges include the inaccessibility of hypoxic niche by therapy-agents and the poor replication of viruses or bacteria intracellular to hypoxic cancer cells. Furthermore, the immune privileged microenvironment of tumor hypoxia could pose a challenge to viral/bacterial-induced immunity against the tumor. In this context, here we have tested a novel stem cell-based approach to deliver BCG infected stem cells to the hypoxic core of tumors. Methods: Human SCC-25 cell line-derived xenograft in NOD/SCID mice exhibit hypoxic zones, where ABCG2+ resides (1). SCC-25 tumor-bearing mice were injected i.v. with CD271+ bone marrow mesenchymal stem cells (BM-MSCs). We recently found that CD271+ BM-MSCs could be infected with M. tuberculosis. The BCG infected CD271+ BM-MSCs were injected to SCC-25 xenograft-bearing mice. The mice were sacrificed on day-20 of stem cell injection and then evaluated for the hypoxic CSCs. Next, we have developed a 4-NQO induced oral cancer cell line, which was injected orthotropic to the tongue of congenic mice. The mice developed tumors, which was hypoxic. In these tumor-bearing mice, BCG-infected murine CD271 BM-MSCs were injected i.v., and animals were monitored for four weeks for tumor growth. On day-20 of injection, mice were sacrificed, and the tumors were collected. In a control group, BCG alone was injected. Results: In both the SCC-25 and an immunocompetent mouse model, we found that BCG or CD271+ BM-MSCs injection alone did not result in marked anti-tumor activity and or elimination of ABCG2+ cells from their hypoxic niche. In contrast, the injection of BCG infected CD271+ BM-MSCs led to marked reduction of tumor growth. Importantly, we observed marked replication of BCG intracellular to CSCs. These results indicate that CD271+ BM-MSCs facilitated the transfer of BCG to cancer cells residing in the hypoxic zone. Importantly, the transfer of BCG was seen more in the immunocompetent model, suggesting potential role of immune system. Conclusions: Our findings indicate that hypoxic CSCs may be targeted by a few BCG infected CD271 BM-MSCs. We propose this could be a novel therapeutic approach to target drug resistance cancer stem cells residing in the hypoxic niche of tumors.1. Bhuyan R et al, Cancer Research, 2016; Volume 76, Issue 14 Supplement, pp. 935. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Seema Bhuyan, Jaishree Garhyan, Hong Li, Rashmi Bhuyan, Herman Yeger, Bikul Das. Targeting oral cancer stem cells in the hypoxic niche by BCG infected mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3903. doi:10.1158/1538-7445.AM2017-3903

Abstract 4020: Identification and characterization of Novel MYC and p53 target molecules from medicinal plants of North East India

Background: Identification and characterization of novel molecular inhibitors that target key regulators of cancer stem cell (CSCs) self-renewal is promising in cancer therapeutics. MYC and p53, the two major cellular transcription factors, acts as key molecular regulators in stem cells and cancer stem cells self-renewal. While MYC enhances, p53 inhibits the self-renewal of stem cells. Thus, developing novel molecules that could potentially target either MYC and or p53 might potential therapeutic application. However, the search for small molecules that could target the self-renewal aspect of these two proteins has been elusive. Previously; we demonstrated that isoprenoid molecule squalene could enhance bone marrow hematopoietic and mesenchymal stem cells (MSCs) by modulating MYC. Importantly, we found that ursolic acid, an isoprenoid molecule found in Tulsi plant, inhibited HIF-1alpha, a transcription factor regulated by MYC and p53. Hence, having these prior experiences on isoprenoids, we speculate that novel isoprenoid may exist that could modulate MYC’s role in cancer self-renewal. We are especially interested in identifying novel isoprenoid molecules (metabolites of mevalonate pathway) found in medicinal plants of North East India, where KaviKrishna laboratory is located. Methods: For the study, we proposed to develop an in vitro self-renewal based assay platform(henceforth known as STEM-TECH platform) to identify novel molecules that act on self-renewal aspect of MYC and p53. In this assay, we subjected the oral cancer cell line SCC-25 to 3-D tumoringeic growth using methylcellulose based method. Using this assay, we have screened 20 herbal extracts of North East India, and selected 3 herbal extracts for further evaluation. For the in vivo study, we added these herbal extracts to drinking water of C57BL/6 mice treated with 4NQO (an oral carcinogen) and FVB/N mice with MYC-induced thymic lymphoma. The mice were observed for 10 weeks and subjected to evaluation of tumor growth, and also evaluation of cancer stem cells using ABCG2, a cell surface marker expressed by cancer stem cells. Results: We found that herbal extracts from Tulsi and Soalu exhibited strong anti-tumor activity in the in vivo mouse models. Importantly, these extracts exhibited the ability to inhibit the self-renewal of MYC driven ABCG2+ cancer cells (1). Also, these extracts were then subjected to in vitro Stem-Tech assay and we found that one herbal extract could modulate the transcriptional binding of MYC. Importantly, we found that squalene, an isoprenoid found in several herbal extracts in NE India could modulate MYC activity by an unknown mechanism. Conclusion: Our results indicate that the vast, untapped herbal plants available in India’s remote North East contain valuable herbal medicinal plant having potential MYC inhibitor. 1. Das B et al. MYC through HIF-2alpha regulates the self-renewal program in cancer stem cells (under review). Note: This abstract was not presented at the meeting. Citation Format: Sora Sandhya, Joyeeta Talukdar, Bidisha Pal, Seema Bhuyan, Debabrat Baishya, Bikul Das. Identification and characterization of Novel MYC and p53 target molecules from medicinal plants of North East India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2017-4020