Prior studies demonstrated that resistance to the ERBB1/2 inhibitor Lapatinib in HCT116 cells was mediated by increased MCL-1 expression. We examined whether inhibition of BCL-2 family function could restore Lapatinib toxicity in Lapatinib adapted tumor cells and enhance Lapatinib toxicity in naive cells. The BCL-2 family antagonist Obatoclax (GX15-070), that inhibits BCL-2/BCL-Xl/MCL-1 function, enhanced Lapatinib toxicity in parental HCT116 and Lapatinib adapted HCT116 cells. In breast cancer lines, regardless of elevated ERBB1/2 expression, GX15-070 enhanced Lapatinib toxicity within 3–12 h.The promotion of Lapatinib toxicity neither correlated with cleavage of caspase 3 nor was blocked by inhibition caspases; and was not associated with changes in the activities of ERK1/2, JNK1/2 or p38 MAPK but with reduced AKT, mTOR and S6K1 phosphorylation. The promotion of Lapatinib toxicity by GX15-070 correlated with increased cytosolic levels of apoptosis inducing factor (AIF) and expression of ATG8 (LC3), and the formation of large vesicles that intensely stained for a transfected LC3-GFP construct. Knockdown of the autophagy regulatory proteins ATG5 or Beclin1 suppressed the induction of LC3-GFP vesicularization and significantly reduced cell killing, whereas knock down of MCL-1 and BCL-Xl enhanced the induction of LC3-GFP vesicularization and significantly enhanced cell killing. Knockdown of Beclin1 and AIF abolished cell killing. Collectively, our data demonstrate that Obatoclax mediated inhibition of MCL-1 rapidly enhances Lapatinib toxicity in tumor cells via a toxic form of autophagy and via AIF release from the mitochondrion.
lapatinib; obatoclax; autophagy; cell death; resistance
The present studies have examined approaches to suppress MCL-1 function in breast cancer cells, as a means to promote tumor cell death. Treatment of breast cancer cells with CDK inhibitors (flavopiridol; roscovitine) enhanced the lethality of the ERBB1 inhibitor lapatinib in a synergistic fashion. CDK inhibitors interacted with lapatinib to reduce MCL-1 expression and overexpression of MCL-1 or knock down of BAX and BAK suppressed drug combination lethality. Lapatinib-mediated inhibition of ERK1/2 and to a lesser extent AKT facilitated CDK inhibitor-induced suppression of MCL-1 levels. Treatment of cells with the BH3 domain/MCL-1 inhibitor obatoclax enhanced the lethality of lapatinib in a synergistic fashion. Knock out of MCL-1 and BCL-XL enhanced lapatinib toxicity to a similar extent as obatoclax and suppressed the ability of obatoclax to promote lapatinib lethality. Pre-treatment of cells with lapatinib or with obatoclax enhanced basal levels of BAX and BAK activity and further enhanced drug combination toxicity. In vivo tumor growth data in xenograft and syngeneic model systems confirmed our in vitro findings. Treatment of cells with CDK inhibitors enhanced the lethality of obatoclax in a synergistic fashion. Overexpression of MCL-1 or knock down of BAX and BAK suppressed the toxic interaction between CDK inhibitors and obatoclax. Obatoclax and lapatinib treatment or obatoclax and CDK inhibitor treatment or lapatinib and CDK inhibitor treatment radiosensitized breast cancer cells. Lapatinib and obatoclax interacted to suppress mammary tumor growth in vivo. Collectively our data demonstrate that manipulation of MCL-1 protein expression by CDK inhibition or inhibition of MCL-1 sequestering function by Obatoclax renders breast cancer cells more susceptible to BAX/BAK-dependent mitochondrial dysfunction and tumor cell death.
MCL-1; Lapatinib; Obatoclax; Flavopiridol; Roscovitine; CDK inhibitor; RTK inhibitor; BCL-2 inhibitor; BAK
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills brain cancer cells. OSU toxicity was enhanced by the HSP90 inhibitor 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) that correlated with reduced expression of ERBB1 and ERBB2. Inhibition of the extrinsic apoptosis pathway blocked the interaction between 17AAG and OSU. OSU toxicity was enhanced by the inhibitor of ERBB1/2/4, lapatinib. Knock down of ERBB1/2/4 in a cell line specific fashion promoted OSU toxicity. Combined exposure of cells to lapatinib and OSU resulted in reduced AKT and ERK1/2 activity; expression of activated forms of AKT and to a lesser extent MEK1 protected cells from the lethal effects of the drug combination. Knock down of PTEN suppressed, and expression of PTEN enhanced, the lethal interaction between OSU and lapatinib. Downstream of PTEN, inhibition of mTOR recapitulated the effects of lapatinib. Knock down of CD95, NOXA, PUMA, BIK or AIF, suppressed lapatinib and OSU toxicity. Knock down of MCL-1 enhanced, and overexpression of MCL-1 suppressed, drug combination lethality. Lapatinib and OSU interacted in vivo to suppress the growth of established tumors. Collectively our data argue that the inhibition of ERBB receptor function represents a useful way to enhance OSU lethality in brain tumor cells.
glioblastoma; medulloblastoma; lapatinib; OSU-03012; apoptosis; autophagy; ERBB1; PTEN
The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.
Sorafenib; Lapatinib; Autophagy; Glioma; AKT; ERK1/2; mTOR; PTEN; p70 S6K; Necrosis
Although ErbB receptors have been implicated in prostate cancer progression, ErbB-directed drugs have not proven effective for prostate cancer treatment. The ErbB3-binding protein EBP1 affects both ErbB2 and androgen receptor signaling, two components of the response to ErbB-targeted therapies. We therefore examined the effects of EBP1 expression on the response to the ErbB1/2 tyrosine kinase inhibitor lapatinib. We found a negative correlation between endogenous EBP1 levels and lapatinib sensitivity in prostate cancer cell lines. We then overexpressed or inhibited expression of EBP1. Silencing EBP1 expression increased lapatinib sensitivity and overexpression of EBP1 increased resistance in androgen-containing media. Androgen depletion resulted in an increased sensitivity of androgen-dependent EBP1 expressing cells to lapatinib, but did not affect the lapatinib sensitivity of hormone resistant cells. However, EBP1 silenced cells were still more sensitive to lapatinib than EBP1-expressing cells in the absence of androgens. The increase in sensitivity to lapatinib following EBP1 silencing was associated with increased ErbB2 levels. In addition, lapatinib treatment increased ErbB2 levels in sensitive cells that express low levels of EBP1, but decreased ErbB2 levels in resistant EBP1-expressing cells. In contrast, ErbB3 and phospho ErbB3 levels were not affected by either changes in EBP1 levels or lapatinib treatment. The production of the ErbB3/4 ligand heregulin was increased in EBP1-silenced cells. EBP1-induced changes in AR levels were not associated with changes in lapatinib sensitivity. These studies suggest that the ability of EBP1 to activate ErbB2 signaling pathways results in increased lapatinib sensitivity.
EBP1; Prostate cancer; ErbB2; Lapatinib
ERBB2/HER2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can promote tumor progression. Breast cancer patients with ERBB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that specifically blocks EGFR/ERBB2 signaling. Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three-dimensional (3D) cultures are decreased under hypoxic conditions. Hypoxia can maintain activation of signaling pathways downstream from ERBB2 including AKT and ERK in the presence of lapatinib. HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions. Under hypoxia, activation of ERK signaling is required for lapatinib resistance as treatment with MEK inhibitor trametinib reverses hypoxia-mediated lapatinib resistance. HIF-1 can bypass the lapatinib-treated inhibition of the ERK pathway via inhibition of the dual-specificity phosphatase 2 (DUSP2). Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance. Thus, our results provide rationale for therapeutic evaluation of the treatment of hypoxic ERBB2 expressing breast tumors with a combination of lapatinib and MEK inhibitors.
breast cancer; hypoxia; HIF-1α; lapatinib; ERBB2/HER2; DUSP2
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies.
In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays.
Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine51 and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser51Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to “rescue” the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser51Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.
These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.
Breast cancer; Lapatinib; Combination therapy; Nck; eIF2-alpha
Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.
EGFR; HER2; HER3; HER4; Herceptin
Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.
ErbB-2; breast cancer; lapatinib; resistance; Src kinase; saracatinib; CXCR4
We set out to study the key effectors of resistance and sensitivity to ErbB2 tyrosine kinase inhibitors, such as lapatinib in ErbB2-positive breast and lung cancers. A cell-based in vitro site-directed mutagenesis lapatinib resistance model identified several mutations, including the gatekeeper ErbB2 mutation ErbB2-T798I, as mediating resistance. ErbB2-T798I engineered cell models indeed show resistance to lapatinib but remain sensitive to the irreversible EGFR/ErbB2 inhibitor, PD168393, suggestive of potential alternative treatment strategies to overcome resistance. Gene expression profiling studies identified a select group of downstream targets regulated by ErbB2 signaling and define PHLDA1 as an immediately downregulated gene upon oncogenic ErbB2 signaling inhibition. We find significant down-regulation of PHLDA1 in primary breast cancer and PHLDA1 is statistically significantly less expressed in ErbB2 negative compared with ErbB2 positive tumors consistent with its regulation by ErbB2. Lastly, PHLDA1 overexpression blocks AKT signaling, inhibits cell growth and enhances lapatinib sensitivity further supporting an important negative growth regulator function. Our findings suggest that PHLDA1 might have key inhibitory functions in ErbB2 driven lung and breast cancer cells and a better understanding of its functions might point at novel therapeutic options. In summary, our studies define novel ways of modulating sensitivity and resistance to ErbB2 inhibition in ErbB2-dependent cancers.
The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance.
Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs.
The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings.
Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.
ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185ErbB2). In addition to p185ErbB2 truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously demonstrated. Here we show that expression of a 95 kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2+ breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2+ breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated c-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib sensitive ErbB2+ breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation, may enhance the clinical efficacy of ErbB2 TKIs.
Truncated; nuclear; ErbB2; resistance; tyrosine kinase inhibitors
Despite the importance of the ErbB2/3 heterodimer in breast cancer progression, the negative regulation of these receptors is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduced both ErbB2 and ErbB3 mRNA and protein levels in human breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. The effect was rapid with a decline in steady state mRNA levels first noted two hours after HRG treatment. HRG reduced the rate of transcription of ErbB2 and ErbB3 mRNA, but did not affect ErbB2 or ErbB3 mRNA stability. To test if ErbB2 kinase activity was required for the HRG-induced downregulation, we treated cells with the ErbB2/EGFR inhibitor lapatinib. Lapatinib diminished the HRG- induced decrease in ErbB2 and ErbB3 mRNA and protein, suggesting that the kinase activity of EGFR/ErbB2 is involved in the HRG-induced receptor down-regulation. Further, HRG-mediated decreases in ErbB2/3 mRNA transcription are reversed by inhibiting the AKT but not MAPK pathway. To examine the functional consequences of HRG-mediated decreases in ErbB receptor levels, we performed cell cycle analysis. HRG blocked cell cycle progression and lapatinib reversed this block. Our findings support a role for HRG in the negative regulation of ErbB expression and suggest that inhibition of ErbB2/3 signaling by ErbB2 directed therapies may interfere with this process.
ErbB2; ErbB3; HRG; HER2; HER3; Breast Cancer
Both erbB3 and IGF-1 receptor (IGF-1R) have been shown to play an important role in trastuzumab resistance. However, it remains unclear whether erbB3- and IGF-1R-initiated signaling pathways possess distinct effects on the sensitivity of lapatinib, a dual tyrosine kinase inhibitor against both EGFR and erbB2, in trastuzumab-resistant breast cancer. Here, we show that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 breast cancer sublines, as compared the parental SKBR3 and BT474 cells, respectively, exhibit refractoriness to lapatinib. Knockdown of erbB3 inhibited Akt in SKBR3-pool2 and BT474-HR20 cells, significantly increased lapatinib efficacy, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. In contrast, specific knockdown of IGF-1R did not alter the cells' responsiveness to lapatinib. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R downregulation, the P-Akt levels remained unchanged. Furthermore, a specific inhibitor of Akt, but not Src, significantly enhanced lapatinib-mediated anti-proliferative/anti-survival effects on SKBR3-pool2 and BT474-HR20 cells. These data indicate that erbB3 signaling is critical for both trastuzumab and lapatinib resistances mainly through the PI-3K/Akt pathway, whereas IGF-1R-initiated Src activation results in trastuzumab resistance without affecting lapatinib sensitivity. Our findings may facilitate the development of precision therapeutic regimens for erbB2-positive breast cancer patients who become resistant to erbB2-targeted therapy.
ErbB3; IGF-1R; lapatinib; trastuzumab; resistance
While the HER2-targeting agents trastuzumab and lapatinib have improved the survival of patients with HER2-positive breast cancer, resistance to these targeted therapies is a major challenge. To investigate mechanisms of acquired lapatinib resistance, we generated acquired lapatinib resistance cell models by extended exposure of two HER2-positive breast cancer cell lines to lapatinib. Genomic and proteomic analyses revealed that lapatinib-resistant breast cancer cells gained additional PI3K activation through activating mutation in PI3K p110α and/or increasing protein expression of existing mutant p110α. p110α protein up-regulation in lapatinib-resistant cells occurred through gene amplification or post-transcriptional upregulation. Knockdown of p110α, but not p110β, the other PI3K catalytic subunit present in epithelial cells, inhibited proliferation of lapatinib-resistant cells, especially when combined with lapatinib. Lapatinib-resistant xenograft growth was inhibited persistently by combination treatment with the p110α-selective PI3K inhibitor BYL719 and lapatinib; the drug combination was also well-tolerated in mice. Mechanistically, the combination of lapatinib plus BYL719 more effectively inhibited Akt phosphorylation and, surprisingly, Erk phosphorylation, than either drug alone in the resistance model. These findings indicate that lapatinib resistance can occur through p110α protein upregulation-mediated, and/or mutation-induced, PI3K activation. Moreover, a combinatorial targeted therapy, lapatinib plus BYL719, effectively overcame lapatinib resistance in vivo and could be further tested in clinical trials. Finally, our findings indicate that p110β may be dispensable for lapatinib resistance in some cases. This allows the usage of p110α-specific PI3K inhibitors and thus may spare patients the toxicities of pan-PI3K inhibition to allow maximal dosage and efficacy.
Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (EGFR, Her-1, or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABCB1 and ABCG2 transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1 or ABCG2 overexpressing cells and inhibited the transport of methotrexate and E217βG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]Iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.
multidrug resistance; ABCB1/P-gp; ABCG2/BCRP/MXR; EGFR tyrosine kinase inhibitor; lapatinib
We determined whether the approved myelofibrosis drug ruxolitinib (Jakafi®), an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could be repurposed as an anti-cancer agent for solid tumors. Ruxolitinib synergistically interacted with dual ERBB1/2/4 inhibitors to kill breast as well as lung, ovarian and brain cancer cells. Knock down of JAK1/2 or of ERBB1/2/3/4 recapitulated on-target drug effects. The combination of (ruxolitinib + ERBB1/2/4 inhibitor) rapidly inactivated AKT, mTORC1, mTORC2, STAT3, and STAT5, and activated eIF2α. In parallel, the drug combination reduced expression of MCL-1, BCL-XL, HSP90, HSP70, and GRP78, and increased expression of Beclin1. Activated forms of STAT3, AKT, or mTOR prevented the drug-induced decline in BCL-XL, MCL-1, HSP90, and HSP70 levels. Over-expression of chaperones maintained AKT/mTOR activity in the presence of drugs and protected tumor cells from the drug combination. Expression of dominant negative eIF2α S51A prevented the increase in Beclin1 expression and protected tumor cells from the drug combination. Loss of mTOR activity was associated with increased ATG13 S318 phosphorylation and with autophagosome formation. Autophagosomes initially co-localized with mitochondria and subsequently with lysosomes. Knock down of Beclin1 suppressed: drug-induced mitophagy; the activation of the toxic BH3 domain proteins BAX and BAK; and tumor cell killing. Knock down of apoptosis-inducing factor (AIF) protected tumor cells from the drug combination, whereas blockade of caspase 9 signaling did not. The drug combination released AIF into the cytosol and increased nuclear AIF: eIF3A co-localization. A 4-day transient exposure of orthotopic tumors to (ruxolitinib + afatinib) profoundly reduced mammary tumor growth over the following 35 days. Re-grown tumors exhibited high levels of BAD S112 phosphorylation and activation of ERK1/2 and NFκB. Our data demonstrate that mitophagy is an essential component of (ruxolitinib + ERBB inhibitor) lethality and that this drug combination should be explored in a phase I trial in solid tumor patients.
ruxolitinib; JAK1/2; afatinib; ERBB1; mitophagy; chaperone; AIF
In the completed phase I trial NCT01450384 combining the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib it was observed that 20 of 33 patients had prolonged stable disease or tumor regression, with one complete response and multiple partial responses. The pre-clinical studies in this manuscript were designed to determine whether [pemetrexed + sorafenib] –induced cell killing could be rationally enhanced by additional signaling modulators. Multiplex assays performed on tumor material that survived and re-grew after [pemetrexed + sorafenib] exposure showed increased phosphorylation of ERBB1 and of NFκB and IκB; with reduced IκB and elevated G-CSF and KC protein levels. Inhibition of JAK1/2 downstream of the G-CSF/KC receptors did not enhance [pemetrexed + sorafenib] lethality whereas inhibition of ERBB1/2/4 using kinase inhibitory agents or siRNA knock down of ERBB1/2/3 strongly promoted killing. Inhibition of ERBB1/2/4 blocked [pemetrexed + sorafenib] stimulated NFκB activation and SOD2 expression; and expression of IκB S32A S36A significantly enhanced [pemetrexed + sorafenib] lethality. Sorafenib inhibited HSP90 and HSP70 chaperone ATPase activities and reduced the interactions of chaperones with clients including c-MYC, CDC37 and MCL-1. In vivo, a 5 day transient exposure of established mammary tumors to lapatinib or vandetanib significantly enhanced the anti-tumor effect of [pemetrexed + sorafenib], without any apparent normal tissue toxicities. Identical data to that in breast cancer were obtained in NSCLC tumors using the ERBB1/2/4 inhibitor afatinib. Our data argue that the combination of pemetrexed, sorafenib and an ERBB1/2/4 inhibitor should be explored in a new phase I trial in solid tumor patients.
pemetrexed; sorafenib; ERBB1; PTEN
In Her2-positive breast cancer patients, inhibition of epidermal growth factor receptor 2 (ErbB2)-signaling is often combined with chemotherapy and radiotherapy. The risk of cardiac toxicity after anthracyclines and radiotherapy is recognized, but little is known about increased risk when these treatments are combined with ErbB2 inhibition. This study investigated whether ErbB2 inhibition increased radiation or anthracycline-induced toxicity. In an in vitro study, human cardiomyocytes were treated with irradiation or doxorubicin, alone or in combination with trastuzumab, and evaluated for cell survival and growth. Groups of mice received 0 or 14 Gy to the heart, alone or in combination with lapatinib, or 3 × 4 mg/kg doxorubicin alone or in combination with lapatinib. Mice were evaluated 40 weeks after treatment for cardiac damage. Changes in cardiac function (99mTc-Myoview gated SPECT) were related to histomorphology and microvascular damage. Radiation or doxorubicin-induced cardiomyocyte toxicity (in vitro) were not exacerbated by trastuzumab. Cardiac irradiation of mice decreased microvascular density (MVD) and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor), but these changes were not exacerbated by lapatinib. Inflammatory responses in the irradiated epicardium (CD45+ and F4/80+ cells) were significantly reduced in combination with lapatinib. Irradiation, doxorubicin, and lapatinib each induced cardiac fibrosis but this was not further enhanced when treatments were combined. At the ultra-structural level, both lapatinib and doxorubicin induced mitochondrial damage, which was enhanced in combined treatments. Lapatinib alone also induced mild changes in cardiac function but this was not enhanced in the combined treatments. Trastuzumab did not enhance direct radiation or anthracycline toxicity of cardiomyocytes in vitro. Lapatinib did not enhance the risk of radiation or anthracycline-induced cardiac toxicity in mice up to 40 weeks after treatment, but mitochondrial damage was more severe after doxorubicin combined with lapatinib.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-013-2707-7) contains supplementary material, which is available to authorized users.
ErbB2; Cardiac microvasculature; Irradiation; Anthracyclines
ERBB2-directed therapy is now a routine component of therapy for ERBB2-amplified metastatic gastroesophageal adenocarcinomas. However, there is little knowledge of the mechanisms by which these tumors develop acquired resistance to ERBB2 inhibition. To investigate this question we sought to characterize cell line models of ERBB2-amplified gastroesophageal adenocarcinoma with acquired resistance to ERBB2 inhibition. We generated lapatinib-resistant (LR) subclones from an initially lapatinib-sensitive ERBB2-amplified esophageal adenocarcinoma cell line, OE19. We subsequently performed genomic characterization and functional analyses of resistant subclones with acquired lapatinib resistance. We identified a novel, acquired SrcE527K mutation in a subset of LR OE19 subclones. Cells with this mutant allele harbour increased Src phosphorylation. Genetic and pharmacologic inhibition of Src resensitized these subclones to lapatinib. Biochemically, Src mutations could activate both the phosphatidylinositol 3-kinase and mitogen activated protein kinase pathways in the lapatinib-treated LR OE19 cells. Ectopic expression of Src
E527K mutation also was sufficient to induce lapatinib resistance in drug-naïve cells. These results indicate that pathologic activation of Src is a potential mechanism of acquired resistance to ERBB2 inhibition in ERBB2-amplified gastroesophageal cancer. Although Src mutation has not been described in primary tumor samples, we propose that the Src hyperactivation should be investigated in the settings of acquired resistance to ERBB2 inhibition in esophageal and gastric adenocarcinoma.
Initial success of inhibitors targeting oncogenes is often followed by tumor relapse due to acquired resistance. In addition to mutations in targeted oncogenes, signaling cross-talks among pathways play a vital role in such drug inefficacy. These include activation of compensatory pathways and altered activities of key effectors in other cell survival and growth-associated pathways.
We propose a computational framework using Bayesian modeling to systematically characterize potential cross-talks among breast cancer signaling pathways. We employed a fully Bayesian approach known as the p1-model to infer posterior probabilities of gene-pairs in networks derived from the gene expression datasets of ErbB2-positive breast cancer cell-lines (parental, lapatinib-sensitive cell-line SKBR3 and the lapatinib-resistant cell-line SKBR3-R, derived from SKBR3). Using this computational framework, we searched for cross-talks between EGFR/ErbB and other signaling pathways from Reactome, KEGG and WikiPathway databases that contribute to lapatinib resistance. We identified 104, 188 and 299 gene-pairs as putative drug-resistant cross-talks, respectively, each comprised of a gene in the EGFR/ErbB signaling pathway and a gene from another signaling pathway, that appear to be interacting in resistant cells but not in parental cells. In 168 of these (distinct) gene-pairs, both of the interacting partners are up-regulated in resistant conditions relative to parental conditions. These gene-pairs are prime candidates for novel cross-talks contributing to lapatinib resistance. They associate EGFR/ErbB signaling with six other signaling pathways: Notch, Wnt, GPCR, hedgehog, insulin receptor/IGF1R and TGF- β receptor signaling. We conducted a literature survey to validate these cross-talks, and found evidence supporting a role for many of them in contributing to drug resistance. We also analyzed an independent study of lapatinib resistance in the BT474 breast cancer cell-line and found the same signaling pathways making cross-talks with the EGFR/ErbB signaling pathway as in the primary dataset.
Our results indicate that the activation of compensatory pathways can potentially cause up-regulation of EGFR/ErbB pathway genes (counteracting the inhibiting effect of lapatinib) via signaling cross-talk. Thus, the up-regulated members of these compensatory pathways along with the members of the EGFR/ErbB signaling pathway are interesting as potential targets for designing novel anti-cancer therapeutics.
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-014-0135-x) contains supplementary material, which is available to authorized users.
Drug resisance; Signaling cross-talk; Bayesian statistical modeling; p1-model; EGFR signaling; Breast cancer; Lapatinib
High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression.
Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT–PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease.
BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase–PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group.
This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.
BCAR4; ERBB2; targeted therapy; breast cancer; tamoxifen resistance
The receptor tyrosine kinase ERBB4, a member of the epidermal growth factor receptor (EGFR) family, is unusual in that when phosphorylated, ERBB4 can undergo intramembrane proteolysis, releasing a soluble intracellular domain (ICD) that activates transcription in the nucleus. We found that ERBB4 activated the transcriptional coactivator YAP, which promotes organ and tissue growth and is inhibited by the tumor-suppressor Hippo pathway. Overexpressing ERBB4 in cultured mammary epithelial cells or adding the ERBB4 ligand neuregulin 1 (NRG1) to breast cancer cell cultures promoted the expression of genes regulated by YAP, such as CTGF. Knocking down YAP or ERBB4 prevented the induction of CTGF expression by NRG1, as did preventing ERBB4 cleavage by treating cells with the pan-EGFR inhibitors lapatinib or erlotinib. A PPxY motif in the ERBB4 ICD enabled its interaction with WW domains in YAP, similar to the mode of interaction between YAP and the kinase LATS1, which inhibits the transcriptional activity of YAP. The ERBB4 ICD coimmunoprecipitated with YAP and TEAD1, a YAP coactivator, suggesting that the ERBB4 ICD may functionally interact with YAP and TEAD to promote transcriptional activity. NRG1 stimulated YAP activity to an extent comparable to that of EGF or LPA (lysophosphatidic acid), known activators of YAP. NRG1 stimulated YAP-dependent cell migration in breast cancer cell lines. These observations connect the unusual nuclear function of a growth factor receptor with a mechanosensory pathway and suggest that NRG1-ERBB4-YAP signaling may underlie the aggressive behavior of tumor cells.
Acquired resistance to anti-EGFR therapy may be due to EGFR-ErbB2 heterodimerization and pathway reactivation. In pre-clinical studies. inhibiting ErbB2 blocked this resistance mechanism and re-sensitized cells to anti-EGFR therapy. Cetuximab targets the EGFR receptor, whereas lapatinib inhibits both EGFR and ErbB2. We conducted a phase I trial to assess the safety, dose-limiting toxicities (DLTS), and maximum-tolerated dose (MTD) of cetuximab and lapatinib in patients with solid tumors.
Patients received standard weekly cetuximab with escalating lapatinib dosages of 750, 1000 or 1250mg daily in 3-week cycles. DLTs were monitored through the end of cycle 2. Pre- and post-treatment tumor biopsies and germ-line DNA were obtained for correlative studies.
Twenty-two patients were enrolled, and 18 each were evaluable for toxicity and response. Fifty-nine percent had prior anti-EGFR therapy. Common toxicities included rash and diarrhea. No patient experienced a DLT at the highest dose level and no grade 4 toxicity was observed. Response included no CRs, 3 PRs, 9 SD, and 6 DP, for an overall response rate of 17% and a clinical benefit rate of 67%. The clinical benefit rate in patients previously treated with anti-EGFR therapy was 70%. Mean treatment duration was 4.7 cycles (range 1–14). Decreased expression of EGFR/ErbB2 pathway components after treatment correlated with response, while increased expression in PI3K, Jak/Stat, and MAPK pathways occurred in non-responders.
The combination of cetuximab and lapatinib was well tolerated, with expected toxicities and notable clinical activity, including in patients with previous anti-EGFR therapy. Further clinical study is warranted.
Phase I; EGFR; ErbB2; cetuximab; lapatinib; clinical trial; solid tumors
Combinatorial targeted therapies are more effective in treating cancer by blocking by-pass mechanisms or inducing synthetic lethality. However, their clinical application is hampered by resistance and toxicity. To meet this important challenge, we developed and tested a novel concept of biomarker-guided sequential applications of various targeted therapies using ErbB2-overexpressing/PTEN-low, highly aggressive breast cancer as our model. Strikingly, sustained activation of ErbB2 and downstream pathways drives trastuzumab resistance in both PTEN-low/trastuzumab-resistant breast cancers from patients and mammary tumors with intratumoral heterogeneity from genetically-engineered mice. Although lapatinib initially inhibited trastuzumab-resistant mouse tumors, tumors by-passed the inhibition by activating the PI3K/mTOR signaling network as shown by the quantitative protein arrays. Interestingly, activation of the mTOR pathway was also observed in neoadjuvant lapatinib-treated patients manifesting lapatinib resistance. Trastuzumab + lapatinib resistance was effectively overcome by sequential application of a PI3K/mTOR dual kinase inhibitor (BEZ235) with no significant toxicity. However, our p-RTK array analysis demonstrated that BEZ235 treatment led to increased ErbB2 expression and phosphorylation in genetically-engineered mouse tumors and in 3-D, but not 2-D, culture, leading to BEZ235 resistance. Mechanistically, we identified ErbB2 protein stabilization and activation as a novel mechanism of BEZ235 resistance, which was reversed by subsequent treatment with lapatinib + BEZ235 combination. Remarkably, this sequential application of targeted therapies guided by biomarker changes in the tumors rapidly evolving resistance doubled the life-span of mice bearing exceedingly aggressive tumors. This fundamentally novel approach of using targeted therapies in a sequential order can effectively target and reprogram the signaling networks in cancers evolving resistance during treatment.
sequential therapy; tumor evolution; targeted therapy; trastuzumab resistance; BEZ235; ErbB2 stabilization