Investigating novel therapies for brain tumors - the roles of MCT4, cellular senescence, and arsenic trioxide treatment

My studies focused on characterizing signaling and metabolic pathways involved in the pathobiology of brain tumors in order to develop improved therapies. One project was centered on investigating oncogenic KIAA1549-BRAF fusion induced senescence as a growth suppressive mechanism in pilocytic astrocytoma. We found that loss of expression of tumor suppressor p16 lead to significantly decreased survival in pilocytic astrocytoma patients. Another major focus was on metabolic targeting in glioblastoma cells. We found that the lactate exporter MCT4 was specifically upregulated under hypoxic conditions in glioblastoma cells, and its overexpression was significantly linked to survival. Silencing MCT4 in glioblastoma neurospheres led to decreased growth in vitro and in vivo, inhibition of clonogenic capacity, and reductions in CD133-positive stem-like tumor cells. This was partially due to an induction of apoptosis. Interestingly, we found that this apoptotic induction and growth inhibition was not due to lack of lactate export, but instead, due to an inhibition of the HIF response, highlighting the importance of aberrant metabolic regulation in cancer. Finally, we investigated the possibility of targeting Notch and Hedgehog signaling simultaneously in glioblastoma neurospheres using arsenic trioxide. We found that aberrant Notch and Hedgehog pathway signaling was decreased following arsenic trioxide treatment, and this decrease was accompanied by decreased neurosphere growth and proliferation, decreased clonogenic capacity, decreased CD133-positive cells, and increased apoptosis. These studies thus identify mechanisms by which several hallmarks of cancer are altered in brain tumors, and our preclinical data suggest some potential therapeutic applications. However, it is important to note that targeting only one or two hallmark changes in cancer often leads to therapeutic resistance, and multimodal therapies will likely be necessary if we are to cure aggressive brain tumors

Interplay between the DNA Damage Response and Immunotherapy Response in Cancer

Genome instability and immune evasion are both defining hallmarks of cancer. Tumorigenesis is frequently initiated when there is DNA damage to a proto-oncogene or tumor suppressor gene and DNA repair mechanisms are lost or insufficient to correct the damage; immune evasion then prevents the host immune system from recognizing these transformed cells. Therapies targeting genomic instability and immune evasion have been effectively used to treat cancer. Genotoxic therapies such as chemoradiation have been employed in cancer treatments for several decades, while immunotherapy is a relatively new class of cancer therapy that has led to disease regression even in patients with advanced cancer. Several recent studies have shown synergy between both classes of therapy targeting these two defining hallmarks of cancer, and different mechanisms are proposed to be involved. Here, we review the different classes of DNA damage, their links to cancer, and their contribution to immunotherapy responses, as well as the different models that are currently being used to study tumor–immune interactions

Hypoxia Promotes Uveal Melanoma Invasion through Enhanced Notch and MAPK Activation

<div><p>The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO₂) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.</p></div

Working model.

<p>The working model shows that under normal oxygen tension GNAQ/GNA11 mutations, present in the G_α subunit of heterotrimeric G proteins (GPCR: G protein-coupled receptors) in the majority of primary uveal melanomas, are responsible for the activation of MAPK pathway under normoxic conditions. MAPK pathway in turn activates mTOR signaling, known to regulate HIF-1α protein translation and tumor growth. In hypoxic conditions, HIF-1α protein levels are further induced by the inhibition of its oxygen-dependent degradation. Such induction leads to Notch1 intracellular domain (NICD1) stabilization and Notch activation. The induction of P-Erk and P-Akt, mediated by a non-canonical Notch signaling, contributes in promoting invasion and metastasis.</p

Genetic inhibition of Notch signaling reduces hypoxia-mediated cellular invasion.

<p><b>A,</b> The protein levels of HIF-1α and CBF1 were determined by Western blot in 92.1 cells infected with CBF1 or scramble shRNAs and exposed to normoxia or hypoxia for 24 hours; β-Actin was used as loading control. <b>B,</b> Growth rate was determined by MTS assay in 92.1 cells infected with CBF1 or scramble shRNAs and exposed to normoxia or hypoxia for 2, 4, 6 days (*p = 0.01; **p = 0.001; ***p<0.0001). <b>C</b>, Transwell invasion assay was performed in 92.1 cells infected with CBF1 or scramble shRNAs and exposed for 24 hours to normoxia or hypoxia (***p<0.0001).</p

Downregulation of HIF-1α reduces cellular invasion in uveal melanoma lines.

<p><b>A, B,</b> HIF-1α protein levels were determined by Western blot in OCM1 (A) and 92.1 cells (B) infected with HIF-1α or scramble shRNAs and exposed to normoxia or hypoxia for 24 hours. <b>C,</b> MTS assays were performed for 2, 4, 7 days in normoxia or hypoxia using OCM1 and 92.1 cells infected with HIF-1α or scramble shRNAs. P values were determined vs scramble control shRNA. <b>D, E,</b> Transwell invasion assay was carried out in OCM1 and 92.1 cells infected with HIF-1α or scramble shRNAs and exposed for 24 hours to normoxia or hypoxia (*p = 0.02; ***p = 0.0003).</p

Hypoxia activates MAPK and Akt pathways.

<p><b>A,</b> Western blot analysis reveals that exposure to hypoxia for 24 hours activates Erk1-2 and Akt proteins in all the uveal melanoma lines, as found using antibodies specific for phospho-Erk1-2^{Thr202/Tyr204} and phospho-Akt^Ser473; total Erk1-2 and Akt were used as loading controls. <b>B</b>, Transwell invasion assay was performed in 92.1 and OCM1 cell lines treated with the Erk1-2 inhibitor SCH772984 at 500 nM, while exposed to normoxia or hypoxia for 24 hours. The microphotographs of the right panels show the invading cells on the lower surface of a Matrigel-coated filter after 24 hours of incubation in normal (21%) or low (1%) oxygen tension in the presence of the Erk1-2 inhibitor (***p<0.0001).</p

Digoxin inhibits cellular invasion in uveal melanoma cells.

<p><b>A,</b> HIF-1α protein levels were determined by Western blot in 92.1 cells exposed to DMSO or digoxin at 100, 300 nM for 24 hours in normoxia or hypoxia. β-Actin was used as loading control. <b>B,</b> MTS growth assays were carried out in 92.1 cells treated with DMSO or digoxin at 100, 300 nM and exposed to normoxia or hypoxia for 3, 5, 7 days (***p<0.0001). <b>C,</b> Transwell invasion assay was performed in 92.1 cells treated with DMSO or digoxin at 100 nM and exposed to normoxia or hypoxia for 24 hours (***p<0.0001). The microphotographs in the right panel show the invading cells on the lower surface of the Matrigel-coated filter after 24 hours of incubation in normal (21%) or low (1%) oxygen tension.</p