Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms

Abstract

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.

Figure 1

Mammary gland-specific expression of PIK3CA^H1047R induces mammary tumors. (a) Generation of a transgenic mouse model expressing HA (haemagglutinin)-tagged human PIK3CA^H1047R under the control of a tetracycline-inducible promoter (TetO). The expression of PIK3CA^H1047R is coupled through an IRES with downstream expression of luciferase. These mice were crossed with MMTV-rtTA (MTB) mice to generate bi-transgenic iPIK3CA^H1047R animals to drive the expression of HAPIK3CA^H1047R in mammary glands. Lower panels demonstrate bioluminescence imaging of iPIK3CA^H1047R mice maintained in the presence or absence of doxycycline. (b) Tumor-free survival curve for iPIK3CA^H1047R mice maintained on doxycycline (n = 81, median tumor free survival 208 days), and three groups of control mice: MTB (n = 12) and tetO-PIK3CA^H1047R (n = 10) mice maintained with doxycycline, and iPIK3CA^H1047R (n = 14) mice maintained in the absence of doxycycyline. All three types of control mice are represented by the blue line. (c) Representative haematoxylin and eosin (H&E)-stained sections of primary mammary tumors from iPIK3CA^H1047R mice subjected to chronic doxycycline treatment. Scale bars, 25 µm. (d) Immunohistochemistry (IHC) for p-AKT(Ser473) and pS6RP(S235/236) performed on tumors isolated from iPIK3CA^H1047R mice maintained on doxycycline (Dox on panels) or 6 days following doxycycline withdrawal (Dox off panels). Representative images are shown. Scale bar, 50 µm. (e) IHC for Ki67 or TUNEL performed on tumors isolated from iPIK3CA^H1047R mice maintained on doxycycline (Dox on panels) or 3 days following doxycycline withdrawal (Dox off panels). Representative images are shown. Scale bar, 50 µm. n = 6 for each group. * P < 0.005 (Student’s t-test).

Figure 2

Tumor responses to doxycycline withdrawal. (a) Primary tumors (135 primary tumors were derived from 107 tumor bearing bi-transgenic mice; 81 mice carried one tumor, 21 mice bore two tumors and 4 mice had three tumors) had three types of response to doxycycline withdrawal. 45 of 135 (33%) regressed completely without re-growth (black bar), 4 of 135 (3%) regressed partially with no re-growth (green bar) and 86 of 135 (64%) regressed partially but then re-grew (red bar). (b) Western blot analyses of HA-p110α^H1047R, p-Akt and p-S6RP in six recurrent tumors (RCT) in the absence of doxycycline and their matched primary tumors (PMT) maintained on doxycycline. Mammary gland tissues from uninduced iPIK3CA^H1047R mice were used as controls. (c) Responses of recurrent tumor transplants to GDC-0941 or vehicle treatment. Data are shown as mean ± S.E.M (n = 6). * P < 0.001 (Student’s t test).

Figure 3

Genetic alterations associated with PIK3CA^H1047R-independent tumor recurrence. Mouse SNP6.0 array analyses of six recurrent tumors identified an amplification region encompassing c-Met in RCT-E565 (a), and a common focal amplification at the c-Myc locus in RCT-D419 and RCT-C658 tumors (b). (c) Western blot analyses of p-Akt (Ser473) and pS6RP(S235/236) in two RCT-E565 xenograft tumors treated with vehicle or PF02341066. Samples were isolated 4 hours after the last dose from mice treated with PF02341066 for 3 days. (d) Responses of RCT-E565 xenograft tumors in NcrNu mice to PF02341066 or vehicle. Data are shown as mean ± S.E.M (each group, n = 6). *P < 0.005, ** P < 0.001 (Student’s t-test).

Figure 4

Elevation of c-Myc drives mammary tumors to become independent of PIK3CA^H1047R and resistant to PI3K inhibition. (a) shRNA knockdown of c-Myc in primary tumor cells isolated from RCT-D419. Western blot analysis of c-Myc in RCT-D419 parental cells or cells infected with the indicated lentiviral shRNAs. Vinculin was used as a loading control. (b) RCT-D419 cells expressing sh-Luc, sh-Myc1 or sh-Myc2 were transplanted into NOD-SCID mice and tumor formation monitored. Downregulation of c-Myc suppressed tumor formation. * P < 0.001 (log-rank test) (c) Western blot analysis of ectopically expressed c-Myc or c-MycT58A in D777 tumor cells isolated from a PIK3CA^H1047R-dependent primary tumor that had been maintained on doxycycline. (d) Bioluminescence imaging showing tumor establishment in NOD-SCID mice transplanted with D777 cells expressing vector, c-MycT58A or c-Myc. These mice were maintained on doxycycline to sustain PIK3CA^H1047R expression. (e) Tumors established by D777 cells expressing a control vector in the presence of doxycycline regressed upon doxycycline withdrawal. Tumors established by D777 cells expressing c-Myc or c-MycT58A continued to grow in the absence of doxycycline. Data are shown as mean ± S.E.M (n = 6). (f) Mice bearing D777-MycT58A tumors were treated with either GDC-0941 or vehicle and tumor growth followed. Data are shown as mean ± S.E.M (n = 6). (g) A schematic diagram summarizing three outcomes of PIK3CA^H1047R-initiated tumors following inactivation of PIK3CA^H1047R expression; tumors either regress (gray box) or recur (red box) via a PI3K pathway-dependent or -independent mechanism. As illustrated, these tumor outcomes affect tumor responses to drug treatment.