AKT promotes epithelial tumorigenesis in the presence and absence of a functional apoptotic pathway
To determine the role of the AKT pathway in epithelial tumorigenesis and the functional relationship with apoptosis, immortalized baby mouse kidney epithelial (iBMK) cells that express BAX/BAK (W2) or that are BAX/BAK-deficient (D3) (Degenhardt et al., 2002b
) were engineered to stably express either a constitutively active form of AKT (myr
-AKT) or RAS (H-rasV12
). Several RAS- and AKT-expressing stable clones were derived from both W2 and D3 parental iBMK cell lines, along with vector controls. All express E1A and p53DD, whereas BAX and BAK are expressed only in W2 (; data not shown; Tan et al., 2005
). To assess the tumorigenic capacity of W2 and D3 cells expressing either activated RAS or AKT, cells were injected subcutaneously into nude mice.
AKT and RAS promote tumor growth in both wild-type and apoptosis-defective genetic backgrounds
The vector control W2 cells (W2 3.1.2, 5, and 6) were poorly tumorigenic with clonal tumor growth occurring beyond 3 months postinjection () (Degenhardt et al., 2002a
; Nelson et al., 2004
; Tan et al., 2005
). Failure of W2 cells to establish tumors efficiently in vivo is attributed to ischemic conditions following implantation in vivo that result in BIM-dependent, BAX/BAK-mediated apoptosis that largely eliminates W2 cells (Nelson et al., 2004
; Tan et al., 2005
). In contrast, RAS rendered W2 cells highly tumorigenic, with tumors forming within 16 days, remarkably faster than BAX/BAK-deficient D3 cells (), where an apoptotic block enables tumor growth within 30–60 days (Degenhardt et al., 2002a
; Nelson et al., 2004
; Tan et al., 2005
). Interestingly, RAS enhanced D3 tumor growth, indicating a tumor-promoting function independent of apoptosis regulation (). AKT promoted W2 tumor growth, although less so than RAS (). In contrast, AKT dramatically enhanced D3 tumor growth to nearly that of RAS (). Although AKT promotes cell survival, AKT-mediated acceleration of tumor growth in BAX/BAK-deficient cells indicates a tumor-promoting activity independent of apoptosis inhibition. Indeed, AKT synergizes with antiapoptotic BCL-xL
to promote leukemogenesis (Karnauskas et al., 2003
) and has prominent roles in regulating cell growth and metabolism that contribute to tumorigenesis (Hanada et al., 2004
). Given the importance of the PI3 kinase/AKT pathway in human tumorigenesis, it was of interest to identify the apoptosis-independent function of AKT that enhanced solid tumor growth observed here.
AKT promotes tumor necrosis associated with metabolic stress when apoptosis is disabled
To examine the mechanism of enhanced tumor growth by AKT, tumors were examined histologically by hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Late-forming tumors derived from W2 cells are invasive carcinomas with a high mitotic rate (P-H3 staining), isolated areas of necrosis, and scattered active caspase-3-positive apoptotic cells (; Figure S1 in the Supplemental Data
available with this article online) (Nelson et al., 2004
). D3 tumors are invasive carcinomas with a high mitotic rate and prevalent polyploid tumor giant cells (; Figure S1
) (Degenhardt et al., 2002a
; Nelson et al., 2004
). Isolated necrotic areas are found, but no apoptosis occurs, as indicated by an absence of active caspase-3 (Figure S1
) (Nelson et al., 2004
). Evidence suggests that ischemic conditions in the tumor microenvironment result in mitotic slippage or adaptation to generate ploidy abnormalities in tumor cells that are preserved by an apoptotic defect (Nelson et al., 2004
AKT enabled W2 cells to form carcinomas with a high mitotic rate without tumor giant cells (; Figure S1
). Surprisingly, and in contrast to the D3 tumors, D3 AKT tumors (and also D3 RAS) were largely necrotic. Viable D3 AKT tumor cells were apparent in a striking growth pattern surrounding blood vessels in layers approximately ten cells deep (). Radiating outward from the regions of viable tumor was a layer of necrotic cells typified by condensed disk-shaped nuclei and eosinophilic (pink) cytoplasm, which were followed by areas of cell debris with possible infiltrating host cells (see below) (). In contrast to D3 tumors that were nearly entirely composed of viable tumor cells, roughly half the volume of D3 AKT and D3 RAS tumors was necrotic. Furthermore, hypoxic conditions localized to necrotic areas of D3 AKT tumors, neither of which were present in D3 tumors (), suggesting that necrosis in vivo was associated with metabolic stress.
IHC for active caspase-3 (Nelson et al., 2004
) indicated scant evidence of apoptosis in D3, D3 AKT, and D3 RAS tumors, although necrotic areas of D3 AKT and D3 RAS tumors showed active caspase-3 positive cells of undetermined origin, possibly tumor or infiltrating host cells (Figure S1
). W2 (Nelson et al., 2004
), W2 AKT, and W2 RAS tumors displayed active caspase-3 scattered throughout tumor tissue (Figure S1
). Furthermore, AKT activation in tumors where apoptosis was inhibited by BCL-2 were necrotic, indicating that promotion of necrosis was independent of the means of apoptosis inactivation (). Thus, activation of AKT or RAS stimulated tumor growth but also activated cell death by necrosis specifically in tumors possessing an apoptotic defect and in association with metabolic stress. Finally, the necrotic tumor phenotype was not merely a reflection of rapid growth, as RAF activation (RAF-CAAX) similarly promoted D3 tumor growth but without necrosis (data not shown). This suggests that AKT activation can profoundly sensitize apoptosis-defective tumors to necrotic cell death.
AKT activation in apoptosis-defective cells stimulates necrosis in response to metabolic stress in vitro
To test the hypothesis that metabolic stress in tumors in vivo was the stimulus for not only apoptotic cell death in W2 cells, but also necrotic cell death in D3 AKT cells, iBMK cell lines were subjected to ischemia in vitro. W2, W2 AKT, D3, and D3 AKT iBMK cell lines were either untreated or incubated in a 1% oxygen gas mixture and no glucose that simulates ischemic conditions in the tumor microenvironment (Nelson et al., 2004
), and cells were analyzed by flow cytometry. While all four cell line genotypes displayed a normal cell cycle profile when untreated, apoptotic cell death occurred, indicated by the increase in the sub-G1 population in treated W2 and W2 AKT cells and activation of caspase-3 (). Although AKT can block apoptosis, this does not occur in ischemia, since AKT requires glucose to inhibit apoptosis (Gottlob et al., 2001
; Plas et al., 2001
). In contrast, apoptosis-defective D3 cells remained viable in ischemia, and cells accumulated at G2/M and beyond () (Nelson et al., 2004
) without activating caspase-3 (). D3 AKT cells, however, lost viability in ischemia due to cellular disintegration without stimulation of caspase-3 activation (). The dramatic induction of cell death by AKT in D3 cells required both glucose and oxygen deprivation, as deprivation of either alone was substantially less effective. This was also observed by trypan blue staining in multiple independent cell lines (data not shown). Electron microscopy (EM) under normal and ischemic conditions revealed apoptotic morphology in ischemic W2 and W2 AKT cells, and necrosis of ischemic D3 AKT cells as indicated by vacuolated cytoplasm () coincident with release of the necrotic marker HMGB1 (Scaffidi et al., 2002
) from nuclei (). Thus, cell death triggered by AKT activation in BAX/BAK-deficient cells under metabolic stress resembles necrosis and not apoptosis, which sensitized these normally death-resistant cells to cell death.
Ischemia induces autophagy in BAX/BAK-deficient iBMK cells that is diverted to necrosis by AKT activation
Strikingly, D3 cells under ischemia displayed a highly unusual ultrastructural morphology consisting of the accumulation of double membrane vesicles containing cytoplasmic organelles indicative of autophagy () while retaining nuclear HMGB1 (). Autophagy can be a method of cell death if allowed to proceed to completion; however, it is also a mechanism for prolonging survival in response to nutrient depletion by permitting the utilization of a cell-internal energy source. This suggested that cells apoptose in response to ischemia, but that defective apoptosis permits sustained autophagy possibly to support survival that may be compromised by AKT activation.
AKT inhibits autophagy-mediated survival in ischemia
To quantitate the level of autophagy induced by ischemia in D3 compared to D3 AKT cells, cytosol to membrane translocation of the autophagy marker EGFP-LC3 was monitored (Mizushima et al., 2004
; Tanida et al., 2004
). Both D3 and D3 AKT cells displayed predominantly diffuse EGFP-LC3 localization under normal growth conditions (). In ischemia there was a dramatic shift in the distribution of EGFP-LC3 from diffuse to punctate localization, indicative of lipidation and membrane translocation in D3 cells that was substantially delayed in D3 AKT cells (). By 48 hr of ischemia, the percentage of translocated EGFP-LC3 in D3 AKT cells approximated that in D3 cells (), but by that time the viability of D3 AKT cells was less than 10% compared to 90% in D3 cells (; data not shown). Similar results were obtained by examining lipidation of endogenous LC3 (data not shown). Thus, in the background of an apoptosis defect, AKT activation impairs autophagy induction in response to metabolic stress, potentially eliminating a survival mechanism resulting in necrosis.
Ischemia induces autophagy in apoptosis-defective cells that when inhibited by AKT promotes cell death by necrosis
Autophagy was also induced by ischemia prior to apoptosis in W2 cells (from 3% to 57% at 6 hr of ischemia), and impaired induction of autophagy was observed in W2 AKT cells (from 10% to 20% at 6 hr of ischemia). In apoptosis-competent cells, starvation induces both autophagy and apoptosis; however, W2 and W2 AKT undergo apoptosis at equal rates (), suggesting that the induction of autophagy does not delay or prevent apoptosis and that apoptosis prevails over autophagy. In contrast to normal cells (cardiac cells under neonatal starvation, for example) where autophagy may delay or prevent apoptosis, oncogene activation predisposes cancer cells to apoptosis that may not allow survival by autophagy.
Knockdown of autophagy promotes sensitivity to metabolic stress
To test if autophagy is the mechanism used by D3 cells to survive ischemia, the expression of the essential autophagy gene beclin1
was knocked down using RNAi, and the impact on cell viability under ischemic conditions was determined. RNAi effectively reduced Beclin1 protein levels relative to the LaminA/C control (), reduced autophagy induction in ischemia (), and impaired survival in ischemia (). Similar results were obtained using RNAi targeting a different sequence in beclin1
or another essential autophagy gene, atg5
). Thus, autophagy enables survival of iBMK cells to metabolic stress when apoptosis is inactivated.
To address if autophagy is generally required for epithelial cancer cells to survive metabolic stress, HeLa cells were engineered with an apoptosis defect through BCL-xL
or BCL-2 expression, and Beclin1 was inhibited using RNAi. Knockdown of Beclin1 () significantly reduced autophagy in metabolically stressed cells with (HeLa) or without (HeLa BCL-xL
or BCL-2) the capacity for apoptosis (). As in D3 cells, knockdown of Beclin1 reduced survival in HeLa cells expressing BCL-xL
or BCL-2 under ischemic conditions in comparison to the LaminA/C RNAi controls. In contrast, Beclin1 knockdown had no effect on the viability of HeLa cells with an intact apoptotic response (). Therefore, the response of epithelial cancer cells to metabolic stress is apoptosis, which, when disabled by BAX/BAK deficiency or by BCL-xL
or BCL-2 expression, permits survival by autophagy. AKT activation, which inhibits autophagy (Arico et al., 2001
) (), or direct downregulation of Beclin1 blocks this survival pathway, stimulating necrotic cell death (). This reliance on autophagy for maintenance of cellular functions during metabolic stress may be further exacerbated by dependency on glycolytic metabolism conferred by AKT.
Necrosis is distinct from apoptosis and is a less efficient means of cell death
The data above illustrate that genetic determinants establish whether cells respond to metabolic stress with apoptosis, autophagy, or necrosis. To begin to compare the result of these three distinct responses to the same stimulus, multifield time-lapse microscopy was used to follow cell fates over 5 days in ischemia in vitro. W2 () and W2 AKT (data not shown) underwent classic apoptosis between 24 and 72 hr characterized by an abrupt morphologic change (within 10 min) with membrane blebbing that was completed in under an hour. This is characteristic of apoptosis and consistent with the apoptotic nuclear morphology and caspase-3 activation (). Cell division in ischemia was limited to less than one prior to the onset of apoptosis (). In contrast, ischemia caused D3 AKT cells to undergo violent cytoplasmic vesicle movement (cytoplasmic boiling) and to become refractile and lyse within 48 hr by a process that was clearly distinguished from apoptosis (). Cell division in ischemic D3 AKT cells was rare, with less than 10% of the cell population dividing prior to the onset of necrosis (). Remarkably, D3 cells surviving ischemia by autophagy continued to proliferate for 72 hr, undergoing several cell divisions, after which proliferation and cell motility slowed to give way to progressive cellular condensation (condensation phase) culminating by 5 days (). However, following condensation, these condensed cells were not dead, as restoration of nutrients and oxygen results in reversal of this process and resumption of proliferation for the majority of cells ().
Distinct morphological features of apoptosis, necrosis, and autophagy in ischemia
To compare the efficiency and extent of the different forms of cell death, W2, W2 AKT, D3, and D3 AKT cells were incubated under ischemic conditions for 3, 5, and 7 days, after which they were returned to normal growth conditions and evaluated for clonogenic survival. Both W2 and W2 AKT cells were killed rapidly and efficiently with survival of less than 1 in 106 cells following 5 days of ischemia, whereas D3 cells remained nearly completely viable even after 7 days in ischemia (). While D3 AKT cells displayed impaired clonogenic survival as expected, necrotic cell death was less efficient and much more asynchronous than apoptosis and resulted in gradual 102- to 103-fold impairment in clonogenic survival ().
Autophagy in tumors localizes to the center prior to acquisition of a blood supply
To determine when and where autophagy occurred during tumorigenesis, tumors generated from D3 cells stably expressing EGFP-LC3 were examined for membrane translocation at 1, 3, and 15 days following implantation in vivo. At days 1 () and 3 (data not shown), EGFP-LC3 localization was diffuse only at the perimeter and was punctate, indicative of autophagy in the center of the tumor mass. Angiogenesis does not occur prior to day 3, and tumors are hypoxic, particularly in interior regions (Nelson et al., 2004
). In contrast, at day 15, by which time D3 tumors have established a blood supply and are no longer hypoxic (Nelson et al., 2004
), the localization of EGFP-LC3 was diffuse, with only scattered rare cells displaying punctate localization (). Thus, autophagy localizes to unvascularized, metabolic stressed regions of tumors. Further conformation of the spatial and temporal occurrence of autophagy in tumors awaits a similar analysis of the beclin1+/+
tumors (see below).
Autophagy in tumors localizes to regions of metabolic stress prior to angiogenesis
Role of beclin1 in oncogene-activated necrosis
To directly address the contribution of autophagy inhibition by AKT to induction of necrosis and stimulation of tumorigenesis, iBMK cells with allelic loss of the essential autophagy gene beclin1 were generated and examined. All beclin1+/− and beclin1+/+ iBMK cell lines expressed E1A and p53DD, and reduced Beclin1 expression was observed as expected in beclin1+/− cell lines (). beclin1+/+ and beclin1+/− iBMK cells were engineered without and with an apoptosis defect (BCL-2 expression, ) to document the impact of a reduced capacity for autophagy on survival and method of cell death.
beclin1 haploinsufficiency impairs survival in ischemia in an apoptosis-defective background
Reduced constitutive autophagy was apparent in beclin1+/−
cells (1%) compared to beclin1+/+
cells (3.5%) even under normal growth conditions as measured by EGFP-LC3 translocation. As with W2 cells, ischemia efficiently induced autophagy in beclin1+/+
cells prior to apoptosis (59% at 6 hr of ischemia), and allelic loss of beclin1
impaired autophagy (18% at 6 hr of ischemia) without altering the time course of apoptosis (). BCL-2 expression blocked apoptosis (), and beclin1
haploinsufficiency diminished constitutive autophagy and its induction in ischemia (). Autophagy induction levels in the BCL-2-expressing cells were robust and similar to BAK/BAK-deficient cells, suggesting that inhibition of Beclin1 by BCL-2 (Pattingre et al., 2005
; Shimizu et al., 2004
) is not a significant factor in these circumstances.
Multiple independent BCL-2-expressing beclin1+/+ and beclin1+/− iBMK cell lines, and representative beclin1+/+ and beclin1+/− cell lines without BCL-2, were incubated in ischemia, and viability was assessed. Both wild-type and beclin1+/− cells were readily killed over the course of 2 days as expected, whereas wild-type cells expressing BCL-2 were markedly resistant, with the number of viable cells increasing in the first 24 hr (). Little effect of beclin1 haploinsufficiency was apparent in cells with an apoptotic response. In the background of an apoptosis defect, however, beclin1 haploinsufficiency diminished the survival advantage provided by BCL-2 (). These findings are consistent with the restoration of cell death in ischemic D3 and HeLa BCL-xL and BCL-2 cells, where Beclin1 and autophagy were compromised by RNAi ().
To extend these observations, beclin1+/+ and beclin1+/− iBMK cell lines expressing BCL-2 (WB-13 and BLNB-12, respectively) were examined over the course of 3 days by time-lapse microscopy. BCL-2 protected wild-type cells from cell death by ischemia () similarly to D3 cells (), which otherwise show substantially reduced viability by 48 hr ( and ). BCL-2 expression in wild-type cells allowed 1 to 2 rounds of cell division in the first 24 hr in ischemia (). Cellular condensation commenced on day 2 and continued through day 3, with the majority of the cells retaining viability (). In contrast, the ability of BCL-2 to provide protection from ischemia was diminished in the beclin1+/− cells. Cell division in ischemic conditions was halted rapidly, with only one in five cells dividing in the first 24 hr, followed by cell death resembling necrosis (). EM revealed condensed but healthy morphology of beclin1+/+ BCL-2 cells, but vacuolated and necrotic morphology of beclin1+/− BCL-2 cells by 7 days in ischemia (), indicative of failed organelle integrity (). Thus, in the background of a defect in apoptosis, autophagy can temporarily sustain homeostasis under conditions of metabolic stress. Cells that are defective for autophagy, however, lack this capacity to insulate themselves from fluctuations in external nutrient availability, which renders them vulnerable to metabolic stress.
Autophagy deficiency promotes tumor growth and necrosis
Haploinsufficiency in beclin1
impairs autophagy and promotes tumorigenesis in mice, and beclin1
is monoallelically deleted in many breast, prostate, and ovarian tumors, indicating a role for autophagy in tumor suppression (Edinger and Thompson, 2003
; Liang et al., 1999
; Qu et al., 2003
; Yue et al., 2003
). Disabling both apoptosis and autophagy in stressed cells may be a formula for induction of necrotic cell death, but whether this is related to the mechanism by which beclin1
haploinsufficiency promotes tumorigenesis is not known. To test the roles of defective apoptosis and autophagy on tumorigenesis, beclin1+/+
iBMK cells without and with BCL-2 were evaluated for tumor growth. beclin1+/+
iBMK cells formed tumors only with prolonged latency (greater than 2 months) () as expected and consistent with clonal emergence (Degenhardt et al., 2002a
; Nelson et al., 2004
; Tan et al., 2005
). Allelic loss of beclin1
slightly accelerated tumor growth compared to beclin1+/+
cells, but tumor growth still took 2 months (), consistent with a modest increase in the frequency of clonal emergence. BCL-2 accelerated tumor growth, which was accelerated even further by allelic loss of beclin1
(). Thus, defective autophagy stimulates tumor growth and synergizes with defective apoptosis to promote tumorigenesis. beclin1+/−
BCL-2 tumor growth was still substantially slower than that of the D3 AKT tumors (), suggesting that inhibition of autophagy by AKT contributes to tumorigenesis but that other AKT functions also play a role in promoting tumor growth. Finally, tumor histology revealed a greater prevalence of necrosis in BCL-2-expressing beclin1+/−
compared to beclin1+/+
tumors (), consistent with the possibility that altering the mode of cell death from apoptosis or autophagy to necrosis can impact tumor growth. Beclin1 haploinsufficient tumors, however, were not as necrotic as tumors with activated AKT, indicating that altered metabolism or growth conferred by AKT may further promote necrosis.
beclin1 haploinsufficiency promotes epithelial tumorigenesis
Tumor necrosis stimulates an inflammatory response
Oncogene-activated necrosis in vivo is associated with enhanced tumor growth. To begin to address the possibility that there is a differential immune response to nonnecrotic and necrotic tumors that may influence tumor growth, D3 and D3 AKT tumors were evaluated for evidence of macrophage infiltration (Mac3 IHC). D3 tumors showed few macrophages, whereas the D3 AKT tumors displayed massive macrophage infiltration throughout all necrotic areas up to the border of healthy tumor tissue (); thus, tumor necrosis is associated with macrophage infiltration. Macrophages and other cells constituting an inflammatory infiltrate produce cytokines and chemokines that impact cell proliferation, angiogenesis, and recruitment of other immune effector cells to the site of a wound, infection, or tumor. Indeed, strong p50 NF-κB IHC staining (both nuclear and cytoplasmic) was observed throughout D3 AKT healthy tumor tissue that was less apparent in D3 tumors (). Furthermore, higher activity of the cytokine and NF-κB-responsive IL-6 promoter-LUC reporter was observed in D3 AKT tumors compared to D3 tumors in vivo (). Thus, oncogene-activated tumor necrosis stimulates the innate immune response that has the potential to impact tumor growth ().
Tumor necrosis is associated with macrophage infiltration and cytokine and chemokine production