Survival Of Hypoxic Tumor Cells Research Articles

Development of potent nanosized carbonic anhydrase inhibitor for targeted therapy of hypoxic solid tumors

Overexpression of two carbonic anhydrase (CA) isoforms, CA IX and XII, in several hypoxic solid tumors provides an extracellular hypoxic microenvironment, interferes with extra- and intracellular pH regulation, thus favoring hypoxic tumor cell survival, proliferation and metastasis. In the current study, a selective inhibitor for human CA isoforms IX and XII (isatin-bearing sulfonamide, WEG-104), was incorporated into nanosized spherical niosomes at high encapsulation efficiency to allow for an enhanced and sustained antitumor activity. In vivo, administration of WEG-104 that is either free (10 mg/kg) or loaded into niosomes (5 mg/kg) into a mice model of Ehrlich ascites solid tumor resulted in comparable efficacy in terms of reduction of tumor weight and volume. Administration of WEG-104-loaded niosomes (10 mg/kg) exhibited superior antitumor activity compared to the free drug, evidenced by reduced tumor weight and volume, marked reduction in the activity of CA IX and XII, and suppression of HIF-1α and MMP-2. Moreover, prominent increase of caspase 3 and pronounced decrease in VEGF immune expression were observed in the treated animals. Hence, loading of molecularly designed compounds that targets CAs in hypoxic solid tumors into nanosized delivery systems provided an auspicious strategy for limiting solid tumor progression and malignancy.

Identifying anal and cervical tumorigenesis-associated methylation signaling with machine learning methods.

Cervical and anal carcinoma are neoplastic diseases with various intraepithelial neoplasia stages. The underlying mechanisms for cancer initiation and progression have not been fully revealed. DNA methylation has been shown to be aberrantly regulated during tumorigenesis in anal and cervical carcinoma, revealing the important roles of DNA methylation signaling as a biomarker to distinguish cancer stages in clinics. In this research, several machine learning methods were used to analyze the methylation profiles on anal and cervical carcinoma samples, which were divided into three classes representing various stages of tumor progression. Advanced feature selection methods, including Boruta, LASSO, LightGBM, and MCFS, were used to select methylation features that are highly correlated with cancer progression. Some methylation probes including cg01550828 and its corresponding gene RNF168 have been reported to be associated with human papilloma virus-related anal cancer. As for biomarkers for cervical carcinoma, cg27012396 and its functional gene HDAC4 were confirmed to regulate the glycolysis and survival of hypoxic tumor cells in cervical carcinoma. Furthermore, we developed effective classifiers for identifying various tumor stages and derived classification rules that reflect the quantitative impact of methylation on tumorigenesis. The current study identified methylation signals associated with the development of cervical and anal carcinoma at qualitative and quantitative levels using advanced machine learning methods.

Nucleus accumbens-associated protein-1 promotes glycolysis and survival of hypoxic tumor cells via the HDAC4-HIF-1\u03b1 axis

Nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the BTB/POZ gene family, has emerging roles in cancer. In this study, we identified the NAC1-HDAC4-HIF-1α axis as an important pathway in regulating glycolysis and hypoxic adaptation in tumor cells. We show that nuclear NAC1 binds to histone deacetylase type 4 (HDAC4), hindering phosphorylation of HDAC4 at Ser246 and preventing its nuclear export that leads to cytoplasmic degradation of the deacetylase. Accumulation of HDAC4 in the nuclei results in an attenuation of HIF-1α acetylation, enhancing the stabilization and transcriptional activity of HIF-1α and strengthening adaptive response of cells to hypoxia. We also show the role of NAC1 in promoting glycolysis in a mouse xenograft model, and demonstrate that knockdown of NAC1 expression can reinforce the antitumor efficacy of bevacizumab, an inhibitor of angiogenesis. Clinical implication of the NAC1-HDAC4-HIF-1α pathway is suggested by the results showing that expression levels of these proteins are significantly correlative in human tumor specimens and associated with the disease progression. This study not only reveals an important function of NAC1 in regulating glycolysis, but also identifies the NAC1-HDAC4-HIF-1α axis as a novel molecular pathway that promotes survival of hypoxic tumor cells.

Abstract 2320: Prox1 marks a stem cell population that promotes tumor progression in intestinal adenomas

Abstract Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of this cell population. We have shown that a subpopulation of cells expressing the Prox1 transcription factor have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth in multiple CRC model systems. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut. To further study the adenoma stem cells in relation to the Prox1-positive cell population, we analyzed Apc-mutant intestinal organoids derived from Prox1-CreER; tdTomatoflox/Stop/flox; Apcmin/+ mice. In this lineage tracing model, the induction of Cre activity results in the expression of the red fluorescent protein tdTomato only in the Prox1-positive adenoma cells. FACS sorted tdTomato-positive cell population displayed a highly elevated capability to form new organoids compared to the tdTomato-negative cells, consistent with stem cells enrichment in the Prox1-positive cell population. Gene expression analysis provided new clues about the identity of the Prox1 positive cells. Citation Format: Zoltan Wiener, Jenny Högström, Ville Hyvönen, Pauliina Kallio, Sarika Heino, Kari Alitalo. Prox1 marks a stem cell population that promotes tumor progression in intestinal adenomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2320. doi:10.1158/1538-7445.AM2015-2320

Bortezomib attenuates HIF-1- but not HIF-2-mediated transcriptional activation.

Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the α subunit of hypoxia-inducible factor (HIF)-1 is not degraded with bortezomib treatment, the heterodimeric HIF-1 fails to transactivate target genes. HIF-1 and HIF-2 are related hypoxia-inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIF-1, but not HIF-2. The present study investigated the effects of bortezomib on HIF-2 activity in cancer cells with different levels of HIF-1α and HIF-2α subunits. HIF-α subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription-polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIF-1-specific target gene; however, it had minimal effects on EPO and GLUT-1, which are target genes of both HIF-1 and HIF-2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF-1, and not HIF-2. This novel finding on the lack of an inhibitory effect of bortezomib on HIF-2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (⩽1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (⩽1% oxygen) and hypoxia mimetic CoCl2. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5mM glucose after 48h of physical hypoxia. However, combining the latter with 100μM CoCl2 was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl2 in 5mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.

Hypoxia and metabolism in cancer.

Interest in targeting metabolism has been renewed in recent years as research increases understanding of the altered metabolic profile of tumor cells compared with that of normal cells. Metabolic reprogramming allows cancer cells to survive and proliferate in the hostile tumor microenvironment. These metabolic changes support energy generation, anabolic processes, and the maintenance of redox potential, mechanisms that are all essential for the proliferation and survival of tumor cells. The metabolic switch in a number of key metabolic pathways is mainly regulated by genetic events, rendering cancer cells addicted to certain nutrients, such as glutamine. In addition, hypoxia is induced when highly proliferative tumor cells distance themselves from an oxygen supply. Hypoxia-inducible factor 1α is largely responsible for alterations in metabolism that support the survival of hypoxic tumor cells. Metabolic alterations and dependencies of cancer cells may be exploited to improve anticancer therapy. This chapter reviews the main aspects of altered metabolism in cancer cells, emphasizing recent advances in glucose, glutamine, and lipid metabolism.

Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas

The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN−/− mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.

Targeting Hypoxic Tumor Cell Viability with Carbohydrate-Based Carbonic Anhydrase IX and XII Inhibitors

Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of which inhibit CA IX and XII within a narrow range of low nanomolar K(i) values (5.3-11.2 nM). We evaluate for the first time carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have potential for development as in vivo cancer cell selective inhibitors.

Hypoxia-Selective Antitumor Agents: Norsesterterpene Peroxides from the Marine Sponge Diacarnus levii Preferentially Suppress the Growth of Tumor Cells under Hypoxic Conditions

As part of an ongoing research program to discover natural products that suppress the hypoxia-activated tumor survival pathways, the lipid extract of the Papua New Guinea marine sponge Diacarnus levii was found to suppress hypoxia-induced HIF-1 activation and hypoxic tumor cell survival. Bioassay-guided isolation of D. levii yielded four new norsesterterpene peroxides, diacarnoxides A - D. Diacarnoxide B exhibits a significantly enhanced ability to suppress the growth of tumor cells under hypoxic conditions.

Fractionated dose of 35-MEV fast neutrons and hypoxic tumor cell survival curve.

The determination of the RBE for the MANTA fast neutrons produced by NRL is inprogress, with the model system using tumor cell population kinetic response patterns assayed in vitro after irradiation in vivo. Ascites tumor cells BW-5147 were irradiated with a clinically usable fast neutron beam from the NRL cyclotron, which is produced by accelrating deuterons to 35 MeV and using htem to bombard a thick berylliumtarget. The comparison of dose-effect relationships was made for doses ranging from30 to 1000 rads. The doses required for an isoeffect on BW-5147 hypoxic tumor cell survival and impairment of its reproductive capacity from fast neutron exposure were not different wheither it was given a single dose or the same dose given in three fractions separated by long recovery periods in situ. No intracellular repair of sublethal injury when the dose was given in three fractions, although the hypoxic BWp5147 tumor cells haveno effective reoxygenation or repopulation in this time interval. The RBE for the fast neutron beam is 4 relative to x rays for fractionated doses at the surviving fractionlevel of 0.6-0.7, while the RBE IS 2.5 FOR SINGLE DOSES. However, at a surviving fraction of 0.1, the RBE is 1.9 for single and 2.8 for fractionated doses. Analysis of thedaily cell population rate or mitotic delay between the two types of radiations at a similiar level of survival.