Mutation of gig and rbf leads to synergistic induction of cell death
In a genetic screen for mutations that modulate the consequence of rbf inactivation, we identified a mutation, 64. While mutation of 64 alone led to large mutant patches in adult eyes, mutation of 64 in conjunction with rbf led to only very small mutant patches (). In addition, adult eyes with rbf,64 double mutant clones were smaller and displayed a rough appearance.
Inactivation of cell death regulators hid or dronc increases the sizes of gig,rbf double mutant clones
The decreased size of rbf,64
double mutant clones could be due to an effect of the two mutations on cell proliferation or cell death. Examination of DNA replication showed no inhibition of proliferation (data not shown). Therefore activated caspase-3 (C3) was used to examine the level of cell death. Consistent with previous reports (Du, 2000
; Moon et al., 2006
), mutation of rbf
led to increased apoptosis near the MF (). Although mutation of 64
alone did not cause significant C3 activation in eye discs, mutation of 64
in conjunction with rbf
mutation significantly expanded the observed apoptosis to both anterior as well as posterior clones in eye discs (, arrows) and increased the overall level of cell death in clones throughout the eye disc (). Furthermore, the synergistic induction of cell death by rbf
mutations is not limited to the eye disc. In wing discs, C3 staining was slightly increased in 64
mutant clones but not in rbf
single mutant clones. Significantly increased C3 staining was observed in rbf,64
double mutant clones (). These results indicate that inactivation of rbf
leads to synergistic induction of cell death in both wing and eye discs.
Synergistic induction of cell death by mutations of rbf and gig
Deficiency mapping revealed that the 64
mutation lies between 76F-77B. The following evidence showed that the 64
mutation is an allele of gig
, the Drosophila
TSC2 homolog: 1) 64
mutation failed to complement a previously identified gig
; 2) the mutant phenotypes of 64
clones in adult eyes are very similar to those of the gig192
clones (); 3) sequencing of the gig
gene in 64
mutant identified a C to T mutation that gives rise to a stop codon after amino acid 431 (Fig. S1
); and 4) rbf,gig192
double mutant clones also show synergistic induction of cell death in both the developing eye and wing discs (Fig. S2
). Therefore we renamed the 64
. Since gig64
does not encode the functional domains of gigas
, it is likely that gig64
constitutes a null allele.
rbf,gig-induced cell death in the anterior and posterior parts of the eye disc exhibit differential requirements for Hid and Dronc
Cell death induced by rbf
single mutant clones is mediated by induction of hid
(Moon et al., 2005
; Tanaka-Matakatsu et al., 2009
). Inactivation of hid
completely abolishes cell death in rbf
mutant clones (Tanaka-Matakatsu et al., 2009
). Interestingly, although mutation of hid
significantly decreased rbf,gig
induced cell death, some level of cell death was still observed in rbf,gig,hid
triple mutant clones, particularly in the anterior of the eye disc (yellow arrows in , Fig. S2G–H
). These observations indicate that cell death induced by mutation of rbf,gig
involve both hid
-dependent and hid
induced cell death is highly dependent on Dronc function (Steele et al., 2009
). While dronc
mutation significantly decreased the death of rbf,gig
mutant cells in the posterior (the differentiating part of the eye disc), inactivation of dronc
has little effect on cell death of rbf,gig
clones in the anterior, the proliferating part of the eye disc (, Fig. S2G–H
). Therefore anterior and posterior rbf,gig
cells displayed a marked difference in their dependence on dronc
for cell death.
Consistent with the observations that inactivation of hid or dronc partially inhibited cell death in rbf,gig mutant clones, inactivation of hid or dronc in conjunction with rbf and gig mutations significantly increased the amount of mutant tissue in adult eyes as well as their overall sizes (). These data also support the idea that that the decreased size of rbf,gig mutant patches in adult eyes is due to the synergistic induction of cell death by rbf and gig.
Cell death induced by rbf,gig mutations requires E2F, S6K, and potentially involves JNK signaling
Cell death induced by rbf
mutation is E2F-dependent (Du, 2000
; Moon et al., 2006
is a de2f1
mutant that encodes a truncated protein missing the C-terminal transactivation and RBF binding domains. dE2F1i2
protein can still dimerize with dDP and bind DNA but is unable to activate transcription or bind RBF (Bosco et al., 2001
). Significantly reduced levels of cell death of rbf,gig
mutant clones were observed in the de2f1i2
background (). In addition, much larger rbf,gig
double mutant clones were observed in adult eyes (). These results indicate that dE2F1 activity is required for the synergistic induction of death of rbf,gig
TSC2 forms a complex with TSC1 to promote GTP hydrolysis by the small GTPase Rheb. TOR (target of rapamycin) encodes a large serine/threonine protein kinase that can be found in two complexes, TORC1 and TORC2. Mutation of gig leads to the accumulation of the GTP-bound Rheb, which induces the activation of TORC1/S6K activity and promotes protein synthesis, metabolism, and cell proliferation. Although mutation of s6k does not block cell death induced by loss of rbf in the MF area, mutation of s6k significantly reduced cell death of rbf,gig mutant cells in both the anterior and the posterior of the developing eye disc () and increased sizes of rbf,gig mutant clones in adult eyes (). These observations show that increased S6K activity is also required for the synergistic induction of cell death of rbf,gig mutants.
The c-Jun N-terminal kinase (JNK) pathway is often involved in eliminating aberrant cells from Drosophila
developing tissues (Igaki, 2009
). Puckered (puc)
, a dual specificity phosphatase, is both a target as well as a negative regulator of JNK signaling in flies. Inhibition of JNK signaling by expressing Puc
significantly decreased cell death in rbf,gig
double mutant cells (), suggesting the potential involvement of JNK stress signaling in the synergistic induction of death of rbf,gig
Knockdown of TSC2 in human cancer cells leads to increased cell death depending on Rb status
Both the Rb/E2F and the TSC2/TOR signaling pathways are highly conserved between flies and mammalian systems. Since Rb is often inactivated in human cancer cells, the observed synergistic cell death induction by inactivation of Rb and TSC2 homologs in Drosophila prompted us to determine if inactivation of TSC2 can specifically induce cell death in Rb mutant cancer cells.
shRNA against the C terminus of TSC2 (shTSC2) was shown to strongly reduce the level of TSC2 (Sun et al., 2008
). Consistent with this, lentivirus containing the shTSC2 construct significantly reduced the endogenous TSC2 levels in both the Rb-mutant DU145 and Rb-WT PC3 prostate cancer cells (). Annexin V and Propidium iodide (PI) staining were used to determine the effect of shTSC2 on cell death. While shTSC2 did not significantly affect cell death under normal culture conditions, significantly elevated levels of cell death were observed in DU145shTSC2
cells but not in control DU145 or PC3shTSC2
cells when cells were cultured under hypoxic conditions (). In addition, the ability of different shTSC2 constructs to induce cell death in DU145 cells was correlated with their ability to decrease the level of TSC2 (Fig. S3A–B
). Furthermore, the increased death of DU145shTSC2
cells is not restricted to hypoxic conditions. Significantly elevated levels of cell death were also observed in DU145shTSC2
but not in DU145control
cells when cells were cultured in low serum (). Consistent with these observations, shTSC2 significantly inhibited proliferation in DU145 cells but not in PC3 cells (). Additionally, increased levels of death in DU145shTSC2
cells were also observed when they were cultured in soft agar (Fig. S3H–I
). Therefore knockdown of TSC2 significantly increased the sensitivity of Rb mutant DU145 cells to death under a variety of stress conditions.
TSC2 knockdown induces cell death in Rb-mutant cancer cells under stress conditions
To demonstrate that shTSC2-induced death in cancer cells is dependent on the absence of Rb function, we determined the effect of expressing WT Rb in DU145shTSC2 cells. Expression of WT Rb protein did not affect the shTSC2-induced decrease in TSC2 protein level () but did significantly decrease shTSC2-induced death in DU145 cells () and partially restored cell proliferation (). Furthermore, knockdown of Rb using shRb in conjunction with shTSC2 in PC3 cells significantly increased cell death () and inhibited cell proliferation (). Taken together, these results demonstrate that cell death induced by shTSC2 is dependent on the absence of Rb function.
Rapamycin was used to determine if shTSC2-induced cell death in DU145 cells depends on TORC1 signaling. Inhibition of TORC1 activity by rapamycin significantly decreased shTSC2-induced cell death ( and Fig. S3L-M
). These results indicate that shTSC2-induced cell death is dependent on increased TORC1 activity. In addition, significantly decreased levels of cell death were observed when Z-VAD was used to inhibit caspase activation ( and Fig. S3J–K
). Therefore shTSC2-induced cell death is largely caspase-dependent.
To determine if shTSC2 can also specifically kill other cancer cells depending on their Rb status, we examined the effect of shTSC2 on Saos-2 (Rb mutant) and MG-63 (Rb WT) osteosarcoma cells as well as MDA-MB-468 (Rb mutant) and MDA-MB-231 (Rb WT) breast cancer cells. shTSC2 significantly reduced TSC2 levels in these different cancer cells (Fig. S3C
). Interestingly, shTSC2 significantly increased death in Rb-mutant Saos-2 and MDA-MB-468 cancer cells but not in Rb-WT MG-63 and MDA-MB-231 cancer cells (Fig. S3D–G
). Therefore, knockdown of TSC2 can induce death in a variety of cancer cells depending on Rb status.
shTSC2 inhibits the growth of Rb mutant cancer cells in soft agar and mouse xenografts
The ability of cancer cells to grow and form colonies in soft agar was used to determine if shTSC2-induced changes in the level of cell death described above correlates with changes in anchorage-independent growth. As shown in , shTSC2 dramatically inhibited the ability of DU145 cells to form colonies in soft agar (). Similarly, while shTSC2 alone did not inhibit PC3 cells from forming colonies in soft agar, shRb in conjunction with shTSC2 did significantly inhibit colony formation (). These results showed that shTSC2-induced cell death in these prostate cancer cells is correlated with the inhibition of cancer cell growth in soft agar.
TSC2 knockdown suppresses anchorage-independent growth of Rb-mutant cancer cells in soft agar and reduces tumor formation in xenograft models
To further assess whether the correlation between shTSC2-induced cell death and inhibition of colony formation can be extended to other cancer cells, the effect of shTSC2 on osteosarcoma and breast cancer cell growth was determined. shTSC2 significantly inhibited colony formation of Rb-mutant cancer cells (Saos-2 and MDA-MB-468) but not Rb-WT cancer cells (MG-63 and MDA-MB-231) (). These results show that shTSC2-induced cell death is correlated with an inhibition of cancer cell growth in soft agar, which is also dependent on the absence of Rb function.
To determine the in vivo
significance of TSC2 knockdown on tumor growth, a mouse xenograft model was used to evaluate the effect of TSC2 knockdown. DU145control
cells were injected into athymic nude mice and tumor growth was followed. While all the mice injected with DU145control
cells had tumor growth, only one tumor formed in mice injected with DU145shTSC2
cells ( and Fig. S4
). Therefore shTSC2 also significantly reduced the incidence of tumor growth in xenograft models. These results, in conjunction with the previous results of increased cell death and inhibition of cell growth in soft agar, suggest that inhibition of TSC2 can potentially be used to specifically target Rb mutant cancer cells.
Overexpression of activated Akt does not inhibit shTSC2-induced cell death
Inactivation of TSC2 leads to the activation of TORC1, which in turn activates S6K (Wullschleger et al., 2006
). S6K has been shown to form a negative feedback loop with IRS proteins that leads to inhibition of Akt signaling (Harrington et al., 2004
; Shah et al., 2004
). Consistent with this, inactivation of TSC2 or TSC1 was shown to activate TORC1 but inhibit TORC2 activity, resulting in the downregulation of Akt signaling (Yang et al., 2006
Western blots using antibodies against total or phospho-Akt (Ser473) were carried out to determine the effect of shTSC2 on Akt activation in DU145 cells. shTSC2 led to reduced phospho-Akt and increased phospho-S6K levels without changing the levels of total Akt or S6K (). Since phosphorylation on Ser473 is required for the full activation of Akt and Akt is known to be an important survival signal, we tested the effect of expressing an activated form of Akt on shTSC2-induced cell death. Expression of activated Akt did not inhibit shTSC2-induced cell death (). These observations suggest that decreased Akt signaling is not likely to be the main cause of cell death in DU145 cells. Since Akt signaling cannot inhibit cell death induced by ROS (Nogueira et al., 2008
; Robey and Hay, 2009
), we investigated the involvement of oxidative stress in shTSC2-induced cell death.
TSC2 knockdown induces ROS generation
Inactivation of Rb and TSC2 synergistically increase oxidative stress
We used DHE, a dye that detects superoxide, to determine if shTSC2 induce oxidative stress in DU145 cells. Highly elevated DHE fluorescence was observed in DU145shTSC2 cells compared to the DU145control cells grown in soft agar (). Similarly, FACS analysis detected significantly elevated DHE fluorescence in DU145shTSC2 cells grown under normal conditions (). Therefore shTSC2 induces significant level of oxidative stress.
Rb WT PC3 cells were used to further characterize the effect of inactivating TSC2 and Rb on ROS induction. In cells grown under normoxia, we found that knockdown of either TSC2 or Rb led to modest but reproducible increases in ROS levels and that knockdown of Rb in conjunction with TSC2 led to further increased ROS levels (). Interestingly, a higher level of ROS was observed in all the treatment groups under hypoxia with the most dramatically increased ROS level observed in PC3shRb+shTSC2 cells (). These observations show that hypoxia increased ROS levels and that shRb and shTSC2 led to a synergistic increase in the level of ROS, which is correlated with increased death of PC3shRb+shTSC2 cells (Compare , , and ).
Similarly, shTSC2 led to a significant increase in ROS levels in DU145 cells, particularly under hypoxic conditions (). Furthermore, while expression of WT Rb alone did not decrease ROS levels, Rb expression significantly decreased shTSC2-induced ROS levels (). These results show that inactivation of Rb and TSC2 synergistically increase ROS levels and that a high level of ROS under hypoxic conditions is correlated with shTSC2-induced death in Rb mutant cancer cells (compare and ).
To test the possibility that stress conditions such as hypoxia might increase the level of oxidative stress above a certain threshold to promote shTSC2-induced cell death, we determined the effect of increasing oxidative stress by addition of H2O2 into the culture media. At concentrations of H2O2 that induced a low level of cell death in DU145control cells, high levels of cell death were observed in DU145shTSC2 cells (). Expression of Rb in DU145shTSC2 cells decreased oxidative stress () and also decreased H2O2-induced cell death (). These results show that DU145shTSC2 cells are much more sensitive to oxidative stress and suggest that stress conditions such as hypoxia contribute to shTSC2-induced cell death, at least in part, by increasing oxidative stress.
shTSC2-induced cell death is mediated by the induction of ROS
Reducing oxidative stress significantly decreases shTSC2-induced cell death and increases cancer cell growth in soft agar
The above observations suggest that cell death induced by inactivation of both Rb and TSC2 are due to synergistically induced oxidative stress. To test this idea, the antioxidant N-acetyl cysteine (NAC) was used to reduce oxidative stress. Addition of NAC significantly reduced ROS levels () and significantly reduced death in DU145shTSC2 cells (). Similarly, NAC treatment also significantly reduced death in PC3shRb+shTSC2 cells (). In addition, reducing oxidative stress by expressing the ROS scavenger enzymes SOD2 or Catalase also significantly decreased the level of shTSC2-induced death in DU145 cells (). Interestingly, expression of Rb in DU145 cells significantly reduced shTSC2 induced ROS and cell death, which is not significantly decreased further by antioxidants (). These observations strongly support the idea that oxidative stress is a critical mediator of cell death in DU145 cells and that Rb plays a critical role regulating ROS when TSC2 is inactivated.
Cell growth in soft agar was used to further determine the effect of reducing oxidative stress on shTSC2-induced inhibition of cancer cell growth. While expression of SOD2 or Catalase did not increase colony growth in DU145control cells, SOD2 or Catalase expression in DU145shTSC2 cells significantly increased colony growth in soft agar (). Similarly, reducing oxidative stress by NAC dramatically increased the growth of DU145shTSC2 but not DU145control cells in soft agar (). Furthermore, NAC treatment also dramatically increased the colony growth of PC3shRb+shTSC2 cells but not the PC3control cells (). In conclusion, these results provide further support for the idea that oxidative stress induced by Rb and TSC2 inactivation contributes to increased cancer cell death and growth inhibition.
Inhibition of protein synthesis reduces shTSC2-induced oxidative stress and cell death
Inhibition of TORC1 by rapamycin significantly inhibited shTSC2-induced cell death ( and Fig. S3L-M
). Since the above results showed that shTSC2-induced cell death is, at least in part, due to increased oxidative stress, we determined the effect of rapamycin on ROS levels. Indeed, rapamycin significantly reduced ROS levels in DU145shTSC2
cells without significantly affecting the ROS levels in control cells (). Since a key function of TORC1 is to stimulate protein synthesis, we determined the effect of inhibiting protein synthesis on shTSC2-induced cell death and oxidative stress. G418 interferes with the function of 80S ribosomes and inhibits protein synthesis in eukaryotic cells. Interestingly, G418 significantly reduced ROS levels in DU145shTSC2
cells but not in DU145control
cells (). Furthermore, while G418 treatment significantly reduced shTSC2-induced cell death (), it did not suppress H2
-induced cell death in DU145 cells (). These results show that the ability of G418 to inhibit shTSC2-induced cell death is correlated with its ability to decrease oxidative stress and suggest that increased protein synthesis contributes to shTSC2-induced oxidative stress and cell death.
SOD2 contributes to Rb inactivation-induced ROS levels and cell death
To investigate how inactivation of Rb induces oxidative stress synergistically with shTSC2, we examined the effect of Rb on ROS scavenger enzyme expression. While the level of SOD2 was very low in control and DU145shTSC2 cells, significantly higher levels of SOD2 were detected when WT Rb was expressed (). Interestingly, under hypoxic conditions shTSC2 led to further increased levels of SOD2 only in the presence of WT Rb (). Therefore, Rb regulates the basal as well as shTSC2-induced SOD2 levels in DU145 cells. The inability of DU145shTSC2 cells to induce SOD2 under hypoxic conditions was correlated with high level of oxidative stress and increased cell death (, ). We also tested the effect of Rb on SOD2 levels in PC3 cells. shTSC2 led to elevated SOD2, particularly under hypoxic conditions (). Interestingly, shRb blocked shTSC2-induced increase in SOD2 levels without affecting the basal SOD2 levels (). The inability of PC3shRb+shTSC2 cells to induce SOD2 is also correlated with high levels of oxidative stress and increased cell death ( and ).
Inactivation of both Rb and TSC2 leads to decreased survival signaling and defective ROS control
To further test the idea that SOD2 contributes to shTSC2-induced cell death, we used shRNA to knockdown SOD2 in PC3 cells (Fig. S5A
). While shTSC2 or shSOD2 alone did not significantly affect cell death in PC3 cells, shTSC2+shSOD2 significantly increased the level of cell death (), which is correlated with significantly increased ROS levels (Fig. S5B
). Furthermore, while expression of Rb significantly inhibited shTSC2-induced cell death in DU145 cells (), similar levels of cell death are observed between the DU145shTSC2
and the DU145shTSC2+Rb+shSOD2
cells (). These results provide strong evidence that SOD2 is a critical target of Rb that contributes to Rb and TSC2 inactivation induced cell death. However, since shRb+shTSC2 induces a higher level of cell death than shSOD2+shTSC2 does in PC3 cells (compare and ), it is likely that additional targets of Rb also contribute to Rb and TSC2 inactivation induced death in these cells.
shTSC2 inhibits de novo lipid synthesis, decreases survival signaling, and induces ER stress
To further characterize the mechanisms that contribute to shTSC2-induced cell death and the acquisition of resistance, we recovered cells from a tumor that developed in a mouse injected with DU145shTSC2
cells. Analysis of these cells showed that the TSC2 protein level was still significantly reduced although not as dramatically as that in DU145shTSC2
cells (Fig. S5C
). However, these cells had regained the ability to grow in soft agar and are resistant to cell death under hypoxia (data not shown). Therefore it appears that these cells have acquired resistance to shTSC2-induced cell death and we refer to these cells as DU145shTSC2-adapt
Microarray experiments were carried out to identify genes that were significantly altered in response to shTSC2 but were restored in DU145shTSC2-adapt
cells. 463 genes showed significant upregulation or downregulation in response to shTSC2. The expression of 170 genes from these 463 was reversed in the adapted cells. These include genes involved in the cell cycle, lipid metabolism, and cell survival signaling such as Bcl-XL and components of the insulin-like growth factor/EGFR/PI3K signaling (Fig. S5D
). The expression of some of the genes was verified by Real-time RT-PCR (). A general agreement was observed between the RT-PCR results and the microarray data. For example, Bcl-XL, the insulin-like growth factor IGF1R, and key enzymes for de novo
lipid synthesis such as ACLY, HMGCS1, and ACACA were all significantly reduced in DU145shTSC2
cells but not in DU145shTSC2-adapt
cells. In fact, the expression of Bcl-XL, HMGCS1, ACACA, and ACLY were actually higher in the adapted cells (). On the other hand, increased Cyclin E expression was detected in DU145shTSC2
cells but not in DU145shTSC2-adapt
cells (). Expression of Bcl-XL strongly inhibited shTSC2-induced cell death () and restored cell growth in soft agar (), suggesting that changes in the expression of Bcl-XL may contribute to shTSC2-induced cell death in DU145shTSC2
cells and the resistance to death in DU145shTSC2-adapt
Cancer cells generally synthesize most of their lipids de novo
. Inhibition of ACL inhibits de novo
lipid synthesis and suppresses tumor cell growth (Hatzivassiliou et al., 2005
; Pearce et al., 1998
). In addition, inhibition of fatty acid or cholesterol synthesis induces cell death in several cancer models (De Schrijver et al., 2003
; Demierre et al., 2005
). To examine the possibility that the decreased expression of genes involved in lipid synthesis may contribute to shTSC2-induced cell death, we examined the effect of inhibiting fatty acid or cholesterol synthesis on ROS levels. Inhibition of fatty acid synthesis by Cerulenin led to increased ROS levels (), consistent with a published report (Migita et al., 2009
). Similarly, inhibition of cholesterol synthesis by Mevastain also increased ROS (). Taken together, these results suggest that inhibition of de novo
lipid synthesis may also contribute to the shTSC2-induced cell death by increasing oxidative stress.
The significantly decreased expression of genes involved in lipid synthesis in conjunction with increased protein synthesis raise the possibility that shTSC2 will induce additional cellular stress in these cells. Indeed increased XBP-1 splicing is observed in DU145shTSC2
cells as compared to the DU145control
cells (), suggesting that ER stress is also induced by shTSC2 in DU145 cells and that reduced level of ER stress is correlated with the resistance of DU145shTSC2-adapt
cells to shTSC2-induced cell death. Activation of ER stress can induce cell death through multiple pathways (Ron and Walter, 2007
). Taken together, our results suggest that inactivation of Rb and TSC2 induces multiple cellular stresses that contribute to synergistic cell death.