Irina A. Vasilevskaya

Autophagy Inhibition Sensitizes Colon Cancer Cells to Antiangiogenic and Cytotoxic Therapy

Purpose: Autophagy is a critical survival pathway for cancer cells under conditions of nutrient or oxygen limitation, or cell stress. As a consequence of antiangiogenic therapy, solid tumors encounter hypoxia induction and imbalances in nutrient supply. We wished to determine the role of autophagy in protection of tumor cells from the effects of antiangiogenic therapy and chemotherapy. We examined the effect of inhibiting autophagy on hypoxic colon cancer cells in vitro and on bevacizumab- and oxaliplatin-treated mouse xenografts in vivo. Experimental Design: The autophagic response to hypoxia and DNA-damaging agents was assessed by fluorescent microscopic imaging, autophagy-related gene expression, and by electron microscopic ultrastructural analysis. Pharmacologic and molecular approaches to autophagy inhibition were taken in a panel of colon cancer cell lines. Mouse xenograft models were treated with combinations of oxaliplatin, bevacizumab, and chloroquine to assess effects on tumor growth reduction and on pharmacodynamic markers of autophagy inhibition. Results: Autophagy was induced in colon cancer models by exposure to both hypoxia and oxaliplatin. Inhibition of autophagy, either with chloroquine or by downregulation of beclin1 or of ATG5, enhanced sensitivity to oxaliplatin under normal and hypoxic conditions in a synergistic manner. Both bevacizumab and oxaliplatin treatments activate autophagy in HT29 murine xenografts. The addition of chloroquine to bevacizumab-based treatment provided greater tumor control in concert with evidence of autophagy inhibition. Conclusions: These findings implicate autophagy as a mechanism of resistance to antiangiogenic therapies and support investigation of inhibitory approaches in the management of this disease. Clin Cancer Res; 19(11); 2995–3007. ©2013 AACR.

Role of Jun and Jun kinase in resistance of cancer cells to therapy

A series of kinases, the mitogen-activated protein (MAP) kinases, serves to regulate cellular responses to various environmental influences in metazoans. Three major pathways have been described, each with some overlap in substrate specificity that causes activation of parallel pathways. The activation of one of these, the Jun kinase pathway, has been implicated in apoptotic responses to DNA damage, cell stress and cytotoxic drugs. Under most circumstances in non-malignant cells it appears that c-Jun N-terminal kinase (JNK) activation is a pro-apoptotic event that results in turn in activation of pro-apoptotic members of Bcl-2 family and cytochrome c release from mitochondria. In cells with dysregulated/mutated proliferation or cell cycle controls, the role of JNK and of c-Jun is more controversial. We distinguish between the transcriptional effects of JNK and other protein interactions in which it participates. The initiation of mitochondrial apoptosis pathways by JNK is independent of its transcriptional effects for the most part. In certain cell types, c-Jun overexpression is clearly a basis for resistance to DNA-damaging drugs, and resistance reversal has been observed using c-jun antisense. This preliminary evidence suggests that c-jun may have a role in drug resistance, but additional work with patient tumor samples is required to validate the potential of the JNK pathway as a target.

Disruption of Signaling through SEK1 and MKK7 Yields Differential Responses in Hypoxic Colon Cancer Cells Treated with Oxaliplatin

Transcriptional changes in response to hypoxia are regulated in part through mitogen-activated protein (MAP) kinase signaling to activator protein 1 (AP-1), and thus contribute to resistance of cancer cells to therapy, including platinum compounds. A key role for JNK in pro-apoptotic signaling in hypoxic cells has previously been established. Here we analyze hypoxic signaling through MAPK kinases to AP-1/c-Jun in the HT29 colon adenocarcinoma cell line, and observe activation of stress-activated pathways mediated predominantly by SEK1 and MKK7. In transient transfection assays, introduction of dominant-negative constructs for both MKK7 and SEK1 abolished hypoxia-induced AP-1 activation. Functional studies of the pathway using HT29-derived cell lines stably expressing mutant SEK1 or MKK7 showed impaired activation of Jun NH2-terminal kinase (JNK) and AP-1 in response to hypoxia, more marked in MKK7-deficient than SEK1-deficient cells. Inhibition of SEK1 rendered hypoxic cells more sensitive to oxaliplatin in vitro, whereas the opposite effect was observed in MKK7-deficient cells. The mutant cell lines grown as mouse xenografts were treated with oxaliplatin, bevacizumab, or both. The SEK1-deficient tumors exhibited greater sensitivity to all treatments, whereas MKK7-deficient cells were resistant in vivo, consistent with in vitro observations. These data support a positive contribution of MKK7/JNK to oxaliplatin cytotoxicity and identify SEK1 as a potential target for reversal of hypoxic resistance to oxaliplatin.

INHIBITION OF JNK SENSITIZES HYPOXIC COLON CANCER CELLS TO DNA DAMAGING AGENTS

Purpose: We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. Experimental Design: In a panel of cell lines, we investigated effects of pharmacologic and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38, and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Results: Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, although synergy is not always hypoxia specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (nonresponsive) lines. In HT29 and SW620 cells, CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, in which tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. Conclusions: These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic. Clin Cancer Res; 21(18); 4143–52. ©2015 AACR.

JNK1 INHIBITION ATTENUATES HYPOXIA-INDUCED AUTOPHAGY AND SENSITIZES TO CHEMOTHERAPY

Inhibition of hypoxia-induced stress signaling through JNK potentiates the effects of oxaliplatin. The JNK pathway plays a role in both autophagy and apoptosis; therefore, it was determined how much of the effect of JNK inhibition on oxaliplatin sensitivity is dependent on its effect on autophagy. We studied the impact of JNK isoform downregulation in the HT29 colon adenocarcinoma cell line on hypoxia- and oxaliplatin-induced responses. Electron microscopic analyses demonstrated that both oxaliplatin- and hypoxia-induced formations of autophagosomes were reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. The role of specific JNK isoforms was defined using HT29-derived cell lines stably expressing dominant-negative constructs for JNK1 and JNK2 (HTJ1.3 and HTJ2.2, respectively). These cell lines demonstrated that functional JNK1 is required for hypoxia-induced autophagy and that JNK2 does not substitute for it. Inhibition of autophagy in HTJ1.3 cells also coincided with enhancement of intrinsic apoptosis. Analysis of Bcl2-family proteins revealed hyperphosphorylation of Bcl-XL in the HTJ1.3 cell line, but this did not lead to the expected dissociation from Beclin 1. Consistent with this, knockdown of Bcl-XL in HT29 cells did not significantly affect the induction of autophagy, but abrogated hypoxic resistance to oxaliplatin due to the faster and more robust activation of apoptosis. Implications: These data suggest that balance between autophagy and apoptosis is shifted toward apoptosis by downregulation of JNK1, contributing to oxaliplatin sensitization. These findings further support the investigation of JNK inhibition in colorectal cancer treatment. Mol Cancer Res; 14(8); 753–63. ©2016 AACR.

Abstract 2267: Inhibition of autophagy in hypoxic HT29 cells reverses hypoxia-induced resistance to oxaliplatin

BACKGROUND: We have shown previously that inhibition of JNK1 renders HT29 colon adenocarcinoma cells more sensitive to hypoxia and/or oxaliplatin due, in part, to impaired autophagy induction. We also showed that the autophagy inhibitor chloroquine sensitizes HT29 to oxaliplatin in vitro and in mouse xenograft model. PURPOSE: Here we investigate further by molecular approaches, including modulation of signaling through JNK, how inhibition of autophagy affects sensitivity of hypoxic colon cancer cells to oxaliplatin. We employed a panel of HT29-derived cell lines: lines stably expressing dominant negative constructs for either JNK1 (HTJ1.3) or JNK2 (HTJ2.2) singly, derivatives of these lines in which the other JNK gene is silenced by viral delivery of shRNA construct (HTJ1s2 and HTJ2s1), as well as HT29-derived lines with down-regulated BclX (HTBX) or Beclin-1 (HTB). RESULTS: We found that down-regulation of JNK1 leads to inhibition of autophagy accompanied by increase in apoptotic (versus necrosis) cell death. Clonogenic assays revealed higher resistance of HTJ1s2 cells to oxaliplatin under hypoxia, whereas HTJ2s1 cells demonstrated slight increase in sensitivity to the drug in this setting. Down-regulation of either Beclin-1 or BclX resulted in significant sensitization to oxaliplatin in oxic and, even more so, in hypoxic conditions (three-folds on average), while exerting differing effects on autophagy induction. CONCLUSIONS: Our data demonstrate that JNK1 plays an important role in autophagy induction under hypoxic conditions in the HT29 colon adenocarcinoma cell line and that its inhibition increases apoptotic cell death in this setting. Inhibition of autophagy induction by down-regulation of JNK1 and knock-down of Beclin-1 or BclX resulted in reversal of oxaliplatin resistance in hypoxic HT29 cells. These findings suggest that targeting these proteins may be of value in colon cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2267. doi:1538-7445.AM2012-2267

Abstract 2872: Induction of autophagy in hypoxic HT29 cells requires functional JNK1

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BACKGROUND: Hypoxic HT29 colon cancer cell lines exhibit higher sensitivity to oxaliplatin when SEK1 function is impaired and higher resistance when MKK7 is down regulated (which in turn, cause more profound inhibition of JNK activation). We observed differential effects of JNK1/JNK2 modulation on oxaliplatin sensitivity of hypoxic HT29 cells to oxaliplatin treatment that implied a pro-survival function for JNK1. Further, we have shown that both oxaliplatin- and hypoxia-induced autophagy in these cells is diminished in the presence of JNK inhibitor SP600125, and that the cells are sensitized thereby. PURPOSE: Here we investigate further how modulation of signaling through JNK influences autophagy in hypoxic HT29 cells treated with oxaliplatin, and expand the cellular model to additional colon cancer cell lines. We created a panel of HT29-derived cell lines stably expressing dominant negative constructs for SEK1 (HTS13), MKK7 (HTM9), JNK1 (HTJ1.3) and JNK2 (HTJ2.2). We also utilized 6 colon cancer cell lines (HCT116, LoVo, RKO1, SW480, DLD1 and HCT15) in which a dominant negative construct for JNK1 was introduced by viral delivery. RESULTS: Our data demonstrate that in HTJ1.3 cells, hypoxic induction of autophagy is impaired. Silencing of JNK2 in these cells by viral delivery of shRNA does not affect either autophagy induction, or sensitivity to oxaliplatin. Downregulation of up-stream activators of JNK, as in the dnSEK1 or dnMKK7 derivatives, similarly does not affect autophagy induction, while still demonstrating differential effects on activation of JNK isoforms and its major target, c-Jun. Finally, we show that colon cancer cell lines differ in their ability initiate autophagy under hypoxia. Correspondingly, the introduction of dominant negative JNK1 causes some enhancement of oxaliplatin cytotoxicity in LoVo, RKO1 and HCT15 cell lines, which all demonstrate a greater propensity to autophagy under these circumstances. CONCLUSIONS: Our data demonstrate that JNK1 plays a central role in autophagy induction under hypoxic conditions. Knockdown of JNK1 renders HT29 cells more sensitive to hypoxia due to impaired autophagy induction, and JNK2 is unable to substitute for JNK1 under these conditions. Our results also imply involvement of alternative JNK1 activators in autophagy induction by hypoxia. Finally, induction of autophagy and enhancement of oxaliplatin cytotoxicity, by this mechanism, appear to be model-specific in colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2872. doi:10.1158/1538-7445.AM2011-2872